SCIENCE CHINA

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•RESEARCH PAPER• September 2021 Vol.64 No.9: 1533–1545 https://doi.org/10.1007/s11427-021-1973-0

MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean

Xiaolong Fang^1†, Xiaoyuan Sun^1†, Xiangdong Yang^2†, Qing Li^3†, Chunjing Lin², Jie Xu⁴, Wenjun Gong¹, Yifan Wang¹, Lu Liu¹, Limei Zhao², Baohui Liu¹, Jun qin^5*, Mengchen Zhang^5*,

Chunbao Zhang^2*, Fanjiang Kong^1*& Meina Li^1*

1Innovative Center of Molecular Genetics and Evolution, School of Life Sciences, Guangzhou University, Guangzhou 510006, China;

2Soybean Research Institute, National Engineering Research Center for Soybean, Jilin Academy of Agricultural Sciences, Changchun 130033, China;

3State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 311401, China;

4Core Facility and Technical Service Center for SLSB, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;

5Cereal & Oil Crop Institute, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang 050031, China Received June 16, 2021; accepted June 29, 2021; published online July 2, 2021

Male sterility is an essential trait in hybrid seed production, especially for monoclinous and autogamous food crops. Soybean male-sterilems1mutant has been known for more than 50 years and could be instrumental in making hybrid seeds. However, the gene responsible for the male-sterile phenotype has remained unknown. Here, we report the map-based cloning and characterization of theMS1gene in soybean.MS1encodes a kinesin protein and localizes to the nucleus, where it is required for the male meiotic cytokinesis after telophase II. We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining. Bothms1and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal, making them perfect tools for producing hybrid seeds.

The identification ofMS1has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.

soybean, male sterility,ms1, kinesin, cytokinesis, hybrid breeding

Citation: Fang, X., Sun, X., Yang, X., Li, Q., Lin, C., Xu, J., Gong, W., Wang, Y., Liu, L., Zhao, L., et al. (2021).MS1is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean. Sci China Life Sci 64, 1533–1545.https://doi.org/10.1007/s11427-021-1973-0

INTRODUCTION

Soybean (Glycine max (L.) Merrill) is one of the world’s most important staple food crops, supplying more than 59%

of plant oil and 70% of vegetable protein for daily consumption, respectively (SoyStats, 2020). The global popu-

lation will increase to 10 billion by 2050, which will challenge the global food supply, especially for protein food (Wu et al., 2014). A substantial increase in soybean yield is urgently needed to ensure global food security. Hybrid breeding is an important approach to increasing the yield and improving the quality of crops. Hybrid vigor increases yield by approximately 15%–50% in corn, rice, wheat, and several other crops (Kim and Zhang, 2018). However, due to soybean’s strong self-pollination and limited germplasm resources to effectively restore fertility, the development of

SCIENCE CHINA Life Sciences

†Contributed equally to this work

*Corresponding authors (Jun qin, email: hbnkydd@163.com; Mengchen Zhang, email: zhangmengchendd@hotmail.com; Chunbao Zhang, email: cbzhang@cjaas.

com; Fanjiang Kong, email:kongfj@gzhu.edu.cn; Meina Li, email:limeina@gzhu.

edu.cn)

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team in 2002 (Sun et al., 1993;Zhao et al., 2004). However, CMS’s genes and underlying molecular mechanisms are still unknown in soybean, which has largely restricted the development of commercial varieties. With the explosion of genomic resources and the rapid development of plant transformation approaches and new gene-editing technologies, more efforts have been invested in identifying genic male sterility (GMS) genes and developing biotechnology- based male-sterility (BMS) systems for hybrid breeding in crops (Tester and Langridge, 2010;Wan et al., 2019).

To date, only 12 male-sterile and female-fertile GMS mutants (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS,msp, andmsNJ) have been identified in soybean and mapped to seven different chromosomes (Nie et al., 2019).

Due to soybean’s complex paleopolyploid genome, laborious tissue culture process and low transformation efficiency, MS4 is the only GMS gene cloned and published; it was identified using a map-based cloning strategy (Thu et al., 2019). Ms4 is homologous to the PHD-finger class of transcription regulators, which is an ortholog of Arabidopsis MALE MEIOCYTE DEATH 1 (MMD1) and required for chromosomal condensation during the microsporogenesis in Arabidopsis; mutations in MMD1 leads to male sterility (Yang et al., 2003b).

The identification and characterization of GMS genes is the key to rapidly establish a sterility system for commercial applications in hybrid soybean production. This study re- ports the cloning, molecular, and cellular biological characterization of the nuclear recessiveMS1locus in soybean.

TheMS1encodes a kinesin protein and localizes to the nucleus. It colocalizes with microtubules (MTs) and functions in male meiotic cytokinesis. The cloned MS1 provides a foundation for large-scale commercial hybrid soybean seed production and will speed up hybrid breeding in soybean.

RESULTS

Phenotypic analysis ofms1mutant

The first male-sterile soybean mutant ms1 resulted from a

growth (Figure S2A in Supporting Information), except the NIL-ms1-2/JL20 produced small and fleshy seedless pods after flowering (Figure 1A and B).

Furthermore, we observed mature pollen grains of the NIL-ms1-2/JL20 plant, which were much larger than those of the fertile NIL-MS1/JL20 plant. Consequently, some of the pollen grains were clustered and dark-stained with I₂-KI (Figure 1C and D). To observe the cytological phenotype in detail, we performed a cross-sectional analysis with anthers from stages 9 to 12 (stage is defined based on the anther development in rice) (Zhang et al., 2011). Tapetal cells un- derwent programmed cell death during these stages, and the middle layer became nearly invisible (Figure 1E). However, the tapetal cells from the NIL-ms1-2/JL20 plants displayed unusual expansion at stages 9 and 10 and started to degen- erate at stage 11; meanwhile, the middle layer of the mutant enlarged quickly (Figure 1F). Additionally, the microspores of the mutant displayed irregular shapes at stages 9 and 10.

They developed into bigger or degradative microspores at stages 11 and 12 (Figure 1F), which is consistent with previous observations that the mutant’s sterility is caused by the failure of cytokinesis after telophase II, leading to a coenocytic microspore (Albertsen and Palmer, 1979).

Positional cloning ofMS1

Classical genetic analysis indicated that the male-sterile character is inherited as a single recessive gene pair (Brim and Young, 1971). Male sterility was evaluated on BC5F2

populations of JL20 andms1-2by I2-KI staining. Segrega- tions were fitted to a theoretical ratio with a chi-square test and matched with the expected 3:1 ratio of fertile and sterile plants with aPvalue of 0.233. These findings revealed that a single recessive gene controls the sterile trait, confirming previous studies. The BC₅F₂segregating populations were used for bulk segregation analysis (BSA) to map the ms1 locus. Two candidate regions significantly associated with male-sterile traits on chromosomes 6 and 13 were identified by the ΔSNP index. We further narrowed the locus to a unique region on chromosome 13 using another BC₅F₂

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population generated with ms1-3as the female parent and Jilin21 (JL21) as the recurrent male parent (Figure S1B in Supporting Information). Finally, thems1locus was initially delimited on chromosome 13 between 20,933,969 and 25,381,253 bp (Figure 2A, Figure S1C and D in Supporting Information), covering the region of the corresponding locus identified previously (Yang et al., 2014).

To further narrow the MS1locus, we conducted a high- resolution linkage analysis using 267 individuals from the BC₅F₂ segregating populations derived fromms1-2and Ji- lin20. Through fine-mapping,MS1was further narrowed to a 182 kb region between M3 and M6 (Figure 2B and Table S1 in Supporting Information). This 182 kb region contained 17 predicted genes according to the Williams 82 reference genome (Figure 2C). Moreover, we compared the genomic sequences of these candidate genes with the BSA-seq data, and found that only one gene (Glyma.13G114200) showed a difference between NIL-MS1/JL20 and NIL-ms1-2/JL20 DNA pools (Table S2 in Supporting Information). Thems1-2 harbored a nucleotide G deletion at 1,470 bp, which caused a frameshift leading to premature termination of Gly- ma.13G114200translation at amino acid 539 (Figure 2D and E). Thems1-3harbored a nucleotide C change to T at 787 bp (Figure 2D and Table S3 in Supporting Information) result- ing in the alteration of a vital amino acid from Arg to Cys in the motor domain (Figure 2E and Figure S3 in Supporting Information).

Confirmation of MS1 identity by new allele and CRISPR/Cas9 technology

To confirm that the dysfunction ofGlyma.13G114200caused thems1male-sterile phenotype, we examined the NIL-ms1- 3/JL21 phenotype in detail. The NIL-ms1-3/JL21 displayed normal vegetative growth compared with NIL-MS1/JL21 (Figure S2B in Supporting Information). Additionally, the fleshy seedless pods and large, dark-stained pollen grains of NIL-ms1-3/JL21 were similar in the NIL-ms1-2/JL20 plant (Figure 3A and B). The presence of sequence variation and changed phenotypes in these two mutants suggested that the Glyma.13G114200gene was a strong candidate for thems1 locus.

To confirm the role of MS1, we mutated Gly- ma.13G114200in Williams 82 through clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genome-editing techni- que (Figure 3C). Two null alleles were obtained and named ms1cr-1 and ms1cr-2, respectively, with 1 bp deletion in ms1cr-1 and 8 bp deletion with 1 bp insertion in ms1cr-2 (Figure 3D). Both of these two mutations caused a frameshift leading to premature termination of translation (Figure 3E).

The tiny pods and large, dark-stained pollen grains produced by the CRISPR/Cas9 knockout mutants were quite similar to those ofms1-2andms1-3mutants (Figure 3F and G). These results demonstrate that Glyma.13G114200 is MS1 and

Figure 1 Phenotypic characterization of thems1mutant. A, Comparison of NIL-MS1/JL20 plants with NIL-ms1-2/JL20 plants at the R5 stage. The NIL- ms1-2/JL20 plant appears as NIL-MS1/JL20, except that the pod development is abnormal since self-pollination does not occur. B, Pods in NIL-MS1/JL20 and NIL-ms1-2/JL20 plants at the R6 stage. C and D, Mature pollen grains of NIL-MS1/JL20 and NIL-ms1-2/JL20 plants stained with I₂-KI (Scale bar:

100 μm). E and F, Transverse section analysis of anthers in NIL-MS1/JL20 and NIL-ms1-2/JL20 plants from stage 9 to stage 12. The red arrow indicates tapetum, the green arrow indicates microspore, and the yellow arrow indicates the middle layer (Scale bar: 50 μm).

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disruption of Glyma.13G114200 function leads to male sterility in soybean.

MS1 encodes a kinesin-like protein with an N-terminal motor domain and a C-terminal NPK1-binding domain (Figure 2). Phylogenetic analysis indicated that Gly- ma.13G114200andGlyma.17G045600are two homologs of NACK2(Figure S4 in Supporting Information) (Nishihama et al., 2002). Therefore, from now on, Glyma.13G114200 and Glyma.17G045600 will be referred to as GmNACK2a and GmNACK2b. Additionally, Glyma.07G096500 and Glyma.07G181200were clustered withNACK1and referred to asGmNACK1aandGmNACK1b(Figure S4 in Supporting Information). Previous studies showed that NACK1 and NACK2regulate somatic and male meiotic cytokinesis, respectively (Tanaka et al., 2004). The phylogenic tree indicated clear divergence between the NACK1 andNACK2 groups (Figure S4 in Supporting Information). More im- portantly, the monocotyledons lost the NACK2 group during the evolutionary process (Figure S4 in Supporting Informa- tion), which implied the differences in meiosis regulation between dicotyledons and monocotyledons. No expression data are available forGmNACK2bin the SoyBase expression database (https://www.soybase.org/sbt/), and we could not

amplify its full-length complementary DNA (cDNA) after trying different reverse transcription enzymes, which suggests that GmNACK2b is possibly a pseudogene. The divergence of NACK2inArabidopsis and soybean indicated that the regulation of male gametogenesis is different in these two species.

MS1is widely expressed and localizes to the nucleus

We next examined the accumulation patterns of the MS1 gene. Quantitative real-time polymerase chain reaction (qRT- PCR) results showed that the MS1gene was expressed in different tissues but was more abundant in tissues containing proliferating cells, such as root, shoot apical meristem, and flowers (Figure 4A). Furthermore, the expression level of MS1 decreased with the process of flower development (Figure 4A). To further reveal the expression pattern ofMS1 during soybean reproductive development, we dissected the flower buds at different developmental stages into various tissues and organs, including anther, filament, stigma, style, ovary, petal and sepal. Results showed that theMS1gene was predominantly expressed in style and ovary other than in the anther at the early stage of flower development (Figure 4B).

Figure 2 Identification of theMS1locus by map-based cloning. A, Physical map of theMS1region based on two independent BSA-seq mappings. B, Fine- mapping of theMS1region on chromosome 13. Thems1locus is mapped to a 182 kb region between the marker M3 and M6 with a segregated population (n=267) from a cross betweenms1-2and JL20. C, The 182 kb region containing 17 predicted genes according to the Williams 82 reference genome. D, Genetic structure of theMS1candidate geneGlyma.13G114200(ms1mutant alleles are indicated). E, Domain structures of MS1 (mutations forms1-2and ms1-3are indicated).

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This suggests that the expression levels of MS1were not positively correlated with its function.

To determine whether the point mutation inms1-2affected the expression level ofMS1, we performed a qRT-PCR assay in the flower buds for NIL-MS1/JL20 and NIL-ms1-2/JL20.

The result showed no significant differences in these two samples at the three developmental stages (Figure 4C).

Likewise, NIL-ms1-3/JL21 showed the same expression pattern as NIL-MS1/JL21 at the three flower developmental stages (Figure S5 in Supporting Information). The expression of MS1 insignificantly reduced in NIL-ms1-2/JL20 when compared with that in NIL-MS1/JL20 in pollen grains (Figure 4D). These results indicated that the two types of point mutations did not change their expressions at the transcriptional level.

To determine the subcellular localization ofMS1, we fused the full-length cDNA to the C-terminus of green fluorescent

protein (GFP). This construct, driven by the constitutive 35S promoter, was transiently expressed inNicotiana benthami- ana leaves. The transient expression assay confirmed the localization ofMS1protein in the nucleus (Figure 4E).

Transcriptome analysis ofms1mutant

To study the role ofMS1in male reproductive development, we performed RNA-seq analysis with anthers selected from NIL-MS1/JL20 and NIL-ms1-2/JL20 plants. The Pearson correlation coefficient demonstrated a high degree of con- sistency among all three biological replicates (Figure 5A).

5,523 genes were identified as differentially expressed genes (DEGs) using cut-offs of fold change (FC)≥2 and q-va- lue≤0.05. Among them, there were 2,834 upregulated and 2,689 downregulated genes (Figure 5B, Tables S4 and S5 in Supporting Information). Subsequently, Gene Ontology

Figure 3 Confirmation ofMS1identity by new allele and CRISPR/Cas9 technology. A, Comparison of pods in a NIL-MS1/JL21 plant with those in a NIL- ms1-3/JL21 plant with all the leaves removed. B, Mature pollen grains of NIL-MS1/JL21 and NIL-ms1-3/JL21 plants stained with I2-KI (Scale bar: 100 μm).

C,MS1knockout mutations generated by CRISPR/Cas9 using a vector containing two tandem targets. Blue lines indicate the target sites of the guide RNAs.

D, The sequences of the twoms1knockout mutants. E, Domain structures of MS1 and the two CRISPR/Cas9 knockout alleles. F, Phenotypes ofms1cr-1, ms1cr-2and WT plants (Williams 82). The phenotype was recorded at the R7 stage. G, Mature pollen grains of WT,ms1cr-1andms1cr-2plants stained with I2-KI (Scale bar: 200 μm).

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(GO) was conducted with these DEGs in biological processes.Figure 5C showed the top 20 most significantly enriched GO terms with the cell wall modification and organization on the top (evaluated byq-value).

For dicotyledons, phragmoplast expansion will lead to the formation of cell walls, also known as cell plates, between the dividing cells. Callose is a unique feature of male meiosis and is deposited between the plasma membrane and cellulose wall, which are later degraded to release the microspores (Echlin and Godwin,1968). The major component of callose isβ-1,3-glucan, which is synthesized by callose synthase and degraded by callase. InArabidopsis, theAtCDM1increased the expression of AtCalS5(callose synthase) but decreased the expression of A6 (callase), and the atcdm1 mutant showed enlarged pollen grains and multinucleate microspores (Lu et al., 2014). Here we found five genes associated with decreased callose synthesis and two genes associated

with increased callose degradation in the ms1-2 mutant (Figure 5D). The abnormal expansion of tapetum during stages 9 and 10 (Figure 1F), and similarly the enlarged pollen grains inatcdm1and soybeanms1mutant (Figures 1 and 3), suggest that the dysfunction ofMS1influences the metabolism of callose and disrupts the cell plate formation.

MS1 colocalizes with microtubules and regulates phragmoplast expansion

The phragmoplast is the structure that serves as a scaffold for cell plate formation between daughter cells. MTs depoly- merize at the center of the phragmoplast, and tubulin monomers are recruited to the leading edge of the phragmoplast. The dynamic changes of MTs drive phragmoplast growth in an inside-out way (Nebenführ and Dixit, 2018). To identify whether MS1 is directly involved in the phragmo-

Figure 4 The expression pattern ofMS1and its protein subcellular localization. A, Relative expression ofMS1in different tissues. R, root; ST, stem; UL, unifoliolate leaf; TL, trifoliate leaf; SAM, shoot apical meristem; EF, early flower bud; BF, flower bud before pollination; AF, flower bud after pollination. B, Relative expression ofMS1in different flower organs at different developmental stages. E, early; B, before pollination; A, after pollination; An, anther; Fi, filament; Sti, stigma; Sty & Ov, style and ovary; Pe, petal; Sep, sepal. C, Relative expression ofMS1in flower buds of NIL-MS1/JL20 and NIL-ms1-2/JL20 plants at different developmental stages. The abbreviations are the same as in (A). D, Relative expression ofMS1in anthers of NIL-MS1/JL20 and NIL-ms1- 2/JL20 plants. E, The subcellular localization of MS1 in tobacco leaves. Green shows the signal from GFP, blue indicates the DAPI-stained nuclei, and aquamarine represents the merged signals from GFP and DAPI (Scale bar: 100 μm). The data for A–D are from three biological replicates (n=3). Student’st- test was used to evaluate thePvalues of the two samples.

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plast expansion, we first examined whether MS1 is localized with MTs. The MS1-GFP fusion protein driven by MS1 native promoter was transformed into pTUB6::mCherry-

TUB6 transgenic Arabidopsisline. The roots of stabilized co-transgenic Arabidopsis plants were used to detect the signal of MS1 and tubulin. These results showed that MS1

Figure 5 Transcriptome analysis of thems1mutant. A, Pearson correlation coefficients between biological replicates of RNA-seq samples.MS1andms1-2 represent NIL-MS1/JL20 and NIL-ms1-2/JL20, respectively. An is short for anther. The anthers from early to unpollinated stages (the day before flowering) were used for RNA-seq. The color indicates the values of the correlation coefficient. B, Numbers of DEGs betweenMS1andms1-2plants. C, Gene Ontology enrichment analysis. The top 20 GO terms of biological processes were shown. Gene Ratio refers to the percentage of DEGs in one GO termvs.total DEGs.

D, Heat maps of DEGs in the cell wall formation and phosphorylation pathways inMS1andms1-2plants. The heat map illustrated the normalized FPKM of gene expression level inMS1andms1-2.

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protein colocalized with MTs (Figure S6 in Supporting In- formation). To reveal the function of MS1 in soybean male gametogenesis, we performed an immunostaining experi- ment with anti-α-tubulin antibodies in NIL-MS1/JL20 and NIL-ms1-2/JL20 male meiocytes. We found that the cell plate properly formed at the tetrad stage in NIL-MS1/JL20 (Figure 6A). However, this was disorganized in NIL-ms1-2/

JL21, which caused the failure of cytokinesis occurrence and formed coenocytic microspores (Figure 6B). These results together suggest that MS1 directly regulated phragmoplast expansion, which subsequently influenced cell plate formation.

DISCUSSION

Compared with the staple food crops, including rice, corn and wheat, the yield of soybean has failed to significantly increase (Liu et al., 2020). Heterosis, also called hybrid vigor, could be one of the potential approaches to boost soybean yield. Soybean is a typical autogamous species, and male sterility provides the most practical way to prevent self- pollination. Recently, biotechnology-BMS systems using genic male-sterile genes have been developed in different crops, such as corn, rice and wheat (Wan et al., 2019;

Whitford et al., 2013). All the genic male-sterile systems are derived from the original design of seed production technology, which was generated to produce recessive GMS lines using the cornmale-sterility 45(ZmMs45) gene, linked with an α-amylase gene to devitalize transgenic pollen and a red fluorescent geneDsRedto mark the transgenic seeds (Wu et al., 2016). The cloning ofMS1makes it possible to generate

the genic male-sterile systems to produce hybrid seeds in soybean. From the perspective of plant breeders, male sterility can also facilitate random mating in a recurrent selection program and aid in introgressing exotic germplasm into elite cultivars. The male-sterile based recurrent selection systems are widely used in soybean breeding programs (Lewers and Palmer, 1997); even though it greatly assisted in intermating, it is still time-consuming and cost-ineffective without knowing the molecular markers associated with the male-sterile locus. Here we developed two molecular markers based on the twoms1alleles, which would identify the male-fertile sibs and only one-fourth of the F₂ plants that would be male-sterile to grow for the next intermating (Figure S7 and Table S1 in Supporting Information).

Controlling male gametogenesis is an area of commercial interest in generating hybrid crops and selective breeding but is also of fundamental biological importance in plant developmental biology. The cloned MS1 provides a tool that can be used to produce hybrid seeds for commercial or research purposes, but there is also a need to highlight the molecular understanding of anther development, which is largely unknown in soybean. The MS1 encodes a kinesin protein belonging to the kinesin-7 subfamily (Lawrence et al., 2004). Kinesins are microtubule-based force-generating proteins, performing many diverse cellular functions including transport of vesicles and organelles, chromosome segregation, microtubule dynamics and morphogenesis (Nebenführ and Dixit, 2018). The kinesin-7 subfamily is greatly expanded in plants, and certain members localize to the cytokinetic apparatus. This is distinct from their animal members and implies their new functions in plant cells (Zhu and Dixit, 2012).

Figure 6 A proposed model forMS1function in soybean fertility (Modified fromSasabe and Machida, 2012). A and B, Microtubule arrays were visualized by immunostaining with anti-a-tubulin antibodies (red) in NIL-MS1/JL20 and NIL-ms1-2/JL20 male meiocytes at the tetrad stage. The nucleus is stained with DAPI (blue). The merged images are shown on the right (Scale bar: 10 μm). C and D, Schematic of male meiosis at telophase II in WT andms1mutant. E, A model showing the functions of MS1 in the phragmoplast formation during cytokinesis in soybean male meiosis. MS1/GmNACK2a interacts with GmMAPKKK and activates the cascade pathway to phosphorylate microtubule-associated protein 65 (GmMAP65). This triggers the dissolution of the anti- parallel overlap microtubules and recruits Golgi-derived vesicles to the mid-zone of the phragmoplast to form a mature cell plate. The MS1/GmNACK2a dependent activation of MAPK cascade is a prerequisite for the expansion of the phragmoplast, which is essential for the formation of the cell plate and cytokinesis.

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The most closely related proteins to soybean MS1 are NACK1/NACK2 in tobacco and AtNACK2/STUD/TET- RASPORE inArabidopsis(Nishihama et al., 2002;Spielman et al., 1997;Tanaka et al., 2004;Yang et al., 2003a). NACK1/

NACK2 were identified as two M-phase-specific kinesin- like proteins in tobacco, named NPK1-activating kinesin (nucleus and phragmoplast-localized protein kinase 1), which colocalizes with NPK1 to increase its kinase activity (Nishihama et al., 2002). The heterologous expression assays indicated that MS1 localized to both the nucleus and MTs (Figure 4; Figure S6 in Supporting Information). This dual localization pattern has also been observed in OsDLK and ZmKIN11 (VKS1) (Huang et al., 2019;Xu et al., 2018). To reveal the significance and mechanisms of the dual localization pattern, we must determine the dynamic changes of MS1 during soybean development in subsequent studies.

Here, we propose a model for MS1 function in soybean fertility (Figure 6C–E). MAP65 belongs to the MT-associated proteins, which help anti-parallel MT binding at the phragmoplast midline (Ho et al., 2011). NACK2 directly interacts and activates the kinase NPK1/ANP, which subsequently induces MPK4 phosphorylation through the mito- gen-activated protein kinase (MAPK) cascade pathway (Sasabe and Machida, 2012). Once MAP65-1 is phos- phorylated by MPK4 (MAPK), it will lose its binding activity, which causes the phragmoplast expansion (Sasabe et al., 2006). The dysfunction of soybean MS1/GmNACK2 blocked the GmMAPKKK cascade pathway, leading to a non-functional GmMAP65 (Figure 6E). Our RNA-seq analysis revealed that the expression of 11 MAPKKK and MAPK genes was altered in thems1-2mutant (Figure 5D).

More interestingly, three NPK1 and three MAP65 homologs in soybean have been identified, and all were downregulated in ms1-2 mutants (Figure 5D, Table S5 in Supporting In- formation), which further supports our proposed model.

TheArabidopsisAtNACK2 is required only for cytokinesis during male gametogenesis, and the atnack2 mutant plants were indistinguishable from wild-type plants during vegetative development (Tanaka et al., 2004). However, so far, all theatnack2mutant lines are partially male-sterile and can produce viable seeds, which differ from the soybeanms1 mutant. One explanation may be functional differentiation, or it may have been simply caused by the weak mutations of the AtNACK2. All the atnack2 alleles are with a T-DNA insertion in the intron or at the end of C-terminals with de- letions in the last few amino acids without destroying the motor domains (Tanaka et al., 2004;Yang et al., 2003a). To distinguish between these two possibilities, we are in the process of generating theAtNACK2CRISPR/Cas9 knockout mutants. The characterization of theMS1gene revealed its conserved function with their homologs inArabidopsisand tobacco.

Male reproductive development in plants involves a series

of events beginning with stamen meristem specification and ending with pollen grain formation and release (Chang et al., 2011;Hackenberg and Twell, 2019). InArabidopsisand rice, genes in the regulatory network of anther and pollen development have been extensively studied (Ma, 2005;Rutley and Twell, 2015; Wilson and Zhang, 2009). Many signaling molecules and key transcription factors have been identified.

The conservation of the regulatory pathways during these processes is demonstrated by the characterization of more than 40 genes controlling male sterility inArabidopsisand rice, respectively (Wan et al., 2019). To date, very little is known about the genes that regulate these processes in soybean. However, the translation of knowledge from model plants helps us speculate such processes in soybean and identify the genes specifically involved in male sterility.

Systematically, documented dynamic changes in gene expression during soybean anther development will provide toolkits and resources for GMS genes in generating hybrid soybean seeds.

Soybean plants carrying either of the twoms1alleles in our studies show complete male-sterile phenotypes in the growth chamber without any natural pollinators, making them perfect gene resources to construct the genic male-sterile systems. A different ms1 allele was also cloned and characterized in recent studies, with a large fragment deletion in male-sterile materials (Jiang et al., 2021;Nadeem et al., 2021). In conclusion, our research will accelerate the development and efficient use of BMS systems for hybrid soybean breeding.

MATERIALS AND METHODS

Plant materials and growth conditions

Soybean cultivars JL20, JL21 and Williams 82; two soybean NIL ofMS1/JL20×ms1-2/JL20 andMS1/JL21×ms1-3/JL21;

ArabidopsisCol-0 ecotype and thepTUB6::mCherry-TUB6 transgenic line andNicotiana benthamianawere used in this study. Arabidopsis and N. benthamiana were grown in a growth chamber under controlled conditions (16 h light/8 h dark, 22°C). Soybean plants were grown in a 25°C growth incubator under long-day (16 h light/8 h dark) conditions or grown in the field at Changchun, China.

BSA sequencing and data analysis

The two NILs were used for BSA sequencing. DNA was extracted according to the manufacturer’s instructions (CWBIO, Beijing, China). Then, DNA from 20 fertile and 20 sterile individuals was selected and mixed equally to generate DNA pools, respectively. Four pools of DNA from the NILs, with the DNA of male parent JL20 and JL21, were subjected to whole-genome sequencing. Genomic DNA was 1541

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letion length polymorphisms (InDels) by the software tool ANNOVAR (Wang et al., 2010). Sliding window analysis was used to calculate the frequency distribution of SNP (SNP-index) in the population of bulked fertile and sterile individuals; SNP-index was determined for all the SNP positions. Δ(SNP-index) value was assessed on the basis of subtraction of the SNP-index between MS1/ms1-2×JL20 and MS1/ms1-3×JL21 DNA pools. The candidate region was identified in these positive or negative peak regions with a 99% confidence interval in 10,000 bootstrap replicates.

Fine-mapping

267 BC5F2individuals derived from the cross ofms1-2and JL20 were used for genotypic and phenotypic tests. Pheno- typic analysis of pollen vitality was examined by I2-KI staining. Additionally, 64 representative male-fertile BC₅F₂ individuals were chosen for the progeny test. Taking together, with the genotypic and phenotypic data of BC₅F₂and BC₅F₃, we delimitedms1locus to a 182 kb region on chromosome 13. The primers used for fine-mapping are listed in Table S1 in Supporting Information.

Vector construction and plant transformation

The pYLCRISPR/Cas9-DB vector was provided by Pro- fessor Yaoguang Liu (Ma et al., 2015) and was reconstructed to pYLCRISPR/Cas9-DBS by adding the spectinomycin resistance gene. Two targets were designed using the CRISPRdirect online tool (http://crispr.dbcls.jp) (Naito et al., 2015) and single-guide RNA (sgRNA) cassettes were generated with U3 and U6 promoters, respectively. The target- sgRNA sequences were then amplified by PCR and introduced into pYLCRISPR/Cas9-DBS vector with Golden Gate ligation using the BsaI cleavage site. The constructed plasmid was introduced into the Agrobacterium strain EHA101 and transformed into the soybean cultivar Williams 82. The primer sequences are listed in Table S1 in Supporting Information.

replicates in each biological sample). F-box gene (Gly- ma.12G051100) was used as an internal control to normalize the gene expression data (Fang et al., 2013). The primer sequences are listed in Table S1 in Supporting Information.

RNA sequencing and data analysis

RNA samples were prepared from anthers collected in the NIL-MS1/JL20 and NIL-ms1-2/JL20 BC₅F₃ plants during the early to after pollination stage. The process of total RNA extraction is described above. After extracting, mRNA was enriched and fragmented, and then reverse transcripted into cDNA with random primers. Subsequently, the cDNA frag- ments were purified with a QiaQuick PCR extraction kit (Qiagen, Netherlands), end-repaired and ligated to sequencing adapters. The cDNA libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnol- ogy Co., Ltd.

Raw reads were filtered by fastp v0.18.0 to get high- quality clean reads (Chen et al., 2018). Paired-end clean reads were used for mapping to the Williams 82 genome with HISAT2.2.4 (Kim et al., 2015). Then the mapped reads of each individual sample were assembled using StringTie v1.3.1 in a reference-based approach (Pertea et al., 2016;

Pertea et al., 2015). For each transcription region, fragment per kilobase of transcript per million mapped reads (FPKM) value was calculated to quantify its expression abundance and variations. Differential expression analysis was conducted by DESeq2 software between NIL-MS1/JL20 and NIL-ms1-2/JL20 (Love et al., 2014). Genes with a parameter of false discovery rate (FDR)≤0.05 and absolute fold change≥2 were filtered as DEGs. Then, DEGs were mapped to GO terms in the GO database (http://www.geneontology.

org/), and gene numbers were calculated for each time.

Significantly enriched GO terms in DEGs compared with the background were defined by a hypergeometric test.

Subcellular localization

The coding regions of MS1/GmNACK2a (Glyma.

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13G114200) were PCR amplified and inserted into the pCAMBIA1302-GFP vector. Then, the construct was transformed intoAgrobacteria tumefaciensGV3101. After cen- trifugation, the overnight culture was resuspended in 2-(N-morpholine)-ethane sulphonic acid (MES) buffer to an A₆₀₀of 0.8, and incubated at room temperature in the dark for 2 h before injecting intoN.benthamiana. GFP fluorescence was assayed after 48 h using an LSM-800 confocal microscope (Zeiss, Germany).

For the overexpression ofMS1in transgenic Arabidopsis with thepTUB6::mCherry-TUB6 marker (Liu et al., 2019), the 1,988 bp of sequence upstream of the MS1 translation initiation site and coding regions was amplified. Then, the fusion sequences were inserted into the pCAMBIA1302- GFP vector (Fang et al., 2021). Subsequently, the vector was introduced into Agrobacteria tumefaciens GV3101 and transformed into the pTUB6::mCherry-TUB6 transgenic lines. GFP and mCherry fluorescence were detected in Arabidopsisroots using an LSM-800 confocal microscope (Zeiss). The primer sequences are listed in Table S1 in Supporting Information.

Immunostaining

The initiating flower buds on top of the inflorescences were fixed in 2% polyformaldehyde for 1 h. Then, the flower buds were digested with enzyme mix (3% cellulase, 3% pectinase, and 0.5% cytohelicase) at 37°C for 1 h. Slides with desired anthers were immersed in wash buffer I (1× PBS, 1% Tri- tonX-100) for 1 h at room temperature and blocked with goat serum (Bosterbio, Shanghai, China) for 30 min. After three rinses with wash buffer II (1× PBS, 0.1% Tween20), slides were incubated with anti-α-tubulin antibody (1:200) (Sigma- Aldrich, USA) overnight at 4°C. Then the slides were washed three times in wash buffer II and blocked with goat serum (Bosterbio) for 1 h. After that, the slides were incubated with secondary antibody Alexa Fluor 594-con- jugated goat 976 anti-mouse (1:200) (Yeasen, Shanghai, China) at 37°C for 1 h. Subsequently, the slides were washed three times in wash buffer II, and then stained with 8-μL DNA stain, 4′6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, UK). Images were captured using Axio Imager A2 fluorescence microscope (Zeiss).

Cytological analysis

For transverse section assays, flower buds at different development stages were fixed in 4% polyformaldehyde in a vacuum for 1 h. The samples were washed with 0.1 mol L⁻¹ PBS (pH 7.2) three times, 20 min each. The samples were then fixed with 1% osmic acid for 1.5 h at 4°C and washed with ddH₂O three times, 20 min each. Subsequently, the samples were dehydrated in a graded ethanol series (30%,

50%, 70%, 80%, 90% and 95%) for 15 min each, and then dehydrated twice in 100% ethanol, 10 min each. The samples were dipped into 1:3, 1:1, 3:1, 5:1 and 1:0 (v/v) embedding medium and acetone for 12 h. The samples were embedded in resin and polymerized at 40°C for 24 h. The anther samples were sliced into 2-μm sections and stained with 0.05%

Toluidine Blue. Images were captured using a fluorescence microscope (Zeiss).

Statistical analysis

The chi-square test was used to determine if segregation ratios deviated from the expected ratios. Student’st-test was used to compare unpaired datasets. All statistical analyses were conducted using SPSS v2.6 (SPSS Inc).

Data availability

Data supporting the findings of this work are available within the paper and its Supplementary Information files. The RNA-seq data supporting this research have been deposited in the NCBI SRA with the accession number PRJNA726823.

Compliance and ethics The author(s) declare that they have no conflict of interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (32072084, 31871648, and 31971969). We gratefully acknowledge Zhaosheng Kong from the Institute of Microbiology, Chinese Academy of Sciences, for sharing the pTUB6::mCherry-TUB6 transgenic Arabidopsis seeds and useful discussions. We would like to thank Dr. Ali Muhammad for editing and improving the manuscript.

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SUPPORTING INFORMATION

The supporting information is available online athttps://doi.org/10.1007/s11427-021-1973-0. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

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