MS5 mediates early meiotic progression and its

MS5 mediates early meiotic progression and its natural variants may have applications for hybrid production in Brassica napus Qiang Xin,a,1 Yi Shen,b,1 Xi Li,a Wei Lu,a,c Xiang Wang,a Xue Han,a Faming Dong,a Lili Wan,a Guangsheng Yang,a Dengfeng Hong,a,2 and Zhukuan Chengb,2

a

National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China b

State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China c

College of Life Science, South-central University for Nationalities, Wuhan 430074, China

1

These persons contribute equally to this work.

2

Corresponding author: Zhukuan Cheng ([email protected]) or Dengfeng Hong ([email protected]).

Short Title MS5 regulates early meiosis progression

Material distribution footnote The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Dengfeng Hong ([email protected]). Phone: 86-27-8728, 1713, Fax: 86-27-8728, 1713



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SYNOPSIS MS5, a novel gene, determines the proper progression of early meiosis, and its different alleles provide a promising pollination-control system for heterosis breeding in Brassica napus.

ABSTRACT During meiotic prophase I, chromatin undergoes dynamic changes to establish a structural basis for essential meiotic events. However, the mechanism that coordinates chromosome structure and meiotic progression remains poorly understood in plants. Here, we characterized a spontaneous sterile mutant MS5bMS5b in oilseed rape (Brassica napus L) and found its meiotic chromosomes were arrested at leptotene. MS5

is

preferentially

expressed

in

reproductive

organs

and

encodes

a

Brassica-specific protein carrying conserved coiled-coil and DUF626 domains with unknown function. MS5 is essential for pairing of homologs in meiosis, but not necessary for the initiation of DNA double-strand breaks. The distribution of the axis element-associated protein ASY1 occurs independently of MS5, but localization of the meiotic cohesion subunit SYN1 requires functional MS5. Furthermore, both the central element of the synaptonemal complex and the recombination element do not properly form in MS5bMS5b mutants. Our results demonstrate that MS5 participates in progression of meiosis during early prophase I and its allelic variants lead to differences in fertility, which may provide a promising strategy for pollination control for heterosis breeding.



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INTRODUCTION During prophase I of meiosis, chromosomes undergo a series of intricate and elaborate changes in configuration that restructure the chromatin into a highly condensed form, ensuring correct segregation of homologous chromosomes and faithful progression of the cell cycle (Zickler and Kleckner, 1999). Chromosomes also pair and synapse with their homologous partners, forming a unique tripartite structure known as the synaptonemal complex (SC). Dynamic SCs provide the structural framework for homologous recombination and the rearrangement of genetic information. Consequently, proper chromosome structure is a prerequisite for the fundamental events of meiosis. In eukaryotic cells, the mechanistically distinct processes of sister chromatid cohesion and chromosome condensation are thought to be the main determinants of higher-order chromosome structure. During meiosis I, cohesins are not only pivotal for establishing and maintaining sister chromatid cohesion, but also act as a structural basis for axial element (AE) formation and SC assembly. In plants, the Arabidopsis thaliana SYN1, the homolog of the meiosis-specific cohesin Rec8, has been verified to play essential roles in chromosome condensation and pairing (Bai et al., 1999; Cai et al., 2003). Rice (Oryza sativa) REC8 and maize (Zea mays) AFD1, homologs of Arabidopsis REC8, are required for elongation of AEs as well as for proper pairing of homologous chromosomes (Golubovskaya et al., 2006; Shao et al., 2011). Condensins belong to another protein complex that plays fundamental roles in chromosome organization in both mitosis and meiosis (Hirano, 2012). Mutations in condensin genes lead to major developmental defects in plants (Liu et al., 2002; Siddiqui et al., 2003; Siddiqui et al., 2006). Although the biological function of condensin in plant meiosis remains elusive, recent studies in Arabidopsis have revealed its role in maintaining normal meiotic chromosome integrity (Schubert et al., 2013; Smith et al., 2014). Chromosome structure and higher-order configuration appear to be closely related to cell cycle progression during meiosis. A typical example is the pachytene checkpoint in yeast, worms, and mice; however, mutations that block chromosome

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synapsis and/or recombination do not appear to induce pachytene arrest in plants. In addition, in mouse spermatocytes, deletion of both REC8 and RAD21L or deletion of either SMC1, SYCP3, SYCP1, or SYCP2 leads to meiotic arrest at the zygotene or early pachytene (Yuan et al., 2000; Revenkova et al., 2004; de Vries et al., 2005; Yang et al., 2006; Herran et al., 2011; Llano et al., 2012). Plants also contain several genes involved in the meiotic cell cycle transition and chromosome structure establishment. The maize null allele afd1-1 loses the canonical morphology of leptotene chromosomes (Golubovskaya et al., 2006). Also in maize, the am1-praI allele causes irreversible arrest in both male and female meiosis at early prophase I (Pawlowski et al., 2009). In rice am1 mutants, microspore mother cells (MMCs) are arrested at leptotene and homologous pairing and synapsis are disrupted, indicating that AM1 is required for the leptotene-zygotene transition (Che et al., 2011). Mutation in rice MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) also leads to arrest in the MMCs during early meiosis (Nonomura et al., 2007). Brassica napus (genome AnAnCnCn) is an allopolyploid species generated from multiple independent, spontaneous hybridizations between ancestor diploids Brassica rapa (ArAr) and Brassica oleracea (CoCo) (Parkin et al., 1995; Chalhoub et al., 2014). Diploid Brassica species, the closest crop plant relatives to the model plant Arabidopsis, diverged from Arabidopsis approximately 13-17 million years ago (Town et al., 2006). Genome sequencing and comparison revealed the existence of conserved genomic blocks between Brassica and Arabidopsis, and orthologous genes in the corresponding conserved blocks generally show high DNA sequence similarity (Liu et al., 2014). Genes involved in meiosis are highly conserved at the protein level across different species. Thus, molecular characterization of meiosis-related genes in B. napus can directly benefit from knowledge in model plants such as Arabidopsis and rice. The successful application of antibodies specific for some hallmark Arabidopsis meiosis proteins to B. napus, including SYN1, ASY1, ZYP1, HEI10, and MLH1, is a good example of this strategy (Grandont et al., 2014). Male-sterile mutants not only serve as good resources for discovering genes

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involved in sexual reproduction, but also provide potential pollination control systems for hybrid production in many crops. Yi3A is a spontaneous genetic male sterile mutant identified in B. napus. Genetic and molecular marker assays demonstrated that the male sterility of Yi3A plants is controlled by a multiallelic gene, MS5, which has three different alleles, i.e., MS5a, MS5b, and MS5c (Lu et al., 2013). Accessions carrying the homozygous MS5b allele display a male-sterile phenotype and are maintained by crossing with the plants homozygous for MS5c. The resulting heterozygous male-sterile plants (MS5bMS5c) serve as the maternal line to produce hybrids by crossing with restorer MS5aMS5a lines. Previous cytological studies showed that anther development in Yi3A plants is arrested at microspore mother cell stage (Yang et al., 1998). The sterility phenotype was also revealed by anther semithin sections in Rs1046A, a male sterile line transferred from Yi3A (Zhou and Bai, 1994; Wan et al., 2010a). However, detail cytological characterization of the meiotic events in Yi3A and its relatives has not been done yet. Here, we describe the map-based cloning of MS5 and reveal its allelic variations leading to the differences in fertility. Using different cytological approaches, we demonstrate that MS5 may act in progression of meiosis by regulating chromosome configuration during early prophase I.

RESULTS Characterization of plant fertility among different MS5 alleles We have previously developed a three-line system based on the MS5 locus containing different alleles in B. napus, including the genetic male-sterile lines FM195A and Rs1046A (hereafter referred to as MS5bMS5b). In the following parts, MS5bMS5b always represents FM195A if there is no specific indication, the maintainer line 7-5 (referred as MS5cMS5c) and the restorer line FM195B (referred as MS5aMS5a). Compared to the well-developed anthers full of pollen grains in MS5aMS5a (Figure 1A and 1F) and MS5cMS5c (Figure 1B and 1G) plants, the anthers in MS5bMS5b and the heterozygous male-sterile plants derived from the cross between

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MS5bMS5b and MS5cMS5c were withered and lacked pollen grains inside (Figure 1C to 1E, 1H to 1J). In addition, we also observed that the seed set was greatly reduced in MS5bMS5b siliques, whether under open or artificial pollination conditions. The number of seeds per silique in MS5bMS5b (1.2 ± 0.7 for FM195A and 4.1 ± 1.2 for Rs1046A, respectively; Figure 1M and 1N) was much less than that in MS5aMS5a (22.5 ± 0.9; Figure 1K) and MS5cMS5c (26.8 ± 1.0; Figure 1L). Interestingly, despite their full male sterility, heterozygous MS5bMS5c plants generated more seeds per silique (18.5 ± 0.8) than the homozygous MS5bMS5b plants (Figure 1O), so the MS5bMS5c plants always served as the male-sterile line for heterosis breeding in oilseed rape. Map-based cloning of MS5 Using the two closest flanking markers (BE10 and SCD8), we previously mapped the MS5c allele to a 21-kb DNA fragment of a bacterial artificial chromosome (BAC) clone (JBnB034L06) on B. napus chromosome A08 (Supplemental Figure 1A and 1B). To reveal the sequence variations of the flanking region among the three MS5 alleles, we further screened a mixed BAC library containing both the MS5a and MS5b alleles with the MS5-linked marker BE10 and other two markers (G3-2 and WKU-2) developed from this region. BE10 amplifies a specific band from the male-sterile line FM195A; this band is 93-bp smaller than that from the restorer FM195B. G3-2 generates a specific PCR product from FM195B but not in FM195A; also, WKU-2 specifically produces a band from FM195A but not in FM195B. Thus, using these markers let us avoid interference from homoeologous copies and identify the target BAC clones covering MS5a (HBnB148J20) and MS5b (HBnB003M09). Sequencing of the two BAC clones showed that the defined regions covering MS5a and MS5b are about 27 kb and 35 kb, respectively, as restricted by BE10 and SCD8, and both carry nine predicted open reading frames (ORFs) (Supplemental Figure 1C and 1D). Sequence alignment showed the MS5a-containing region has a perfect collinearity with that of MS5b except for two insertion/deletions, while both of them exhibit obvious sequence differences with MS5c (Supplemental Figure 1B to 1D).

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Further comparative mapping with the B. napus reference genome indicated that the predicted ORFs of JBnB034L06 (except for ORF2 and ORF3) are located on chromosome A08 with the highest similarity (Supplemental Figure 1A and 1B) (Chalhoub et al., 2014). However, ORFs of both HBnB148J20 and HBnB003M09 are anchored to two homeologous chromosomes: members from ORF5ʹ to ORF9ʹ mapped to chromosome A08, while the others from ORF1ʹ to ORF4ʹ mapped to chromosome C08 (Supplemental Figure 1). These results suggest that a homeologous chromosome exchange might have happened between the region from BnaA08g25920D to BnaA08g25930D on A08 and the region from BnaC08g14080D to BnaC08g14090D on C08 in the progenitors of FM195A and FM195B. Sequence analysis revealed that only BnaA08g25920D, which corresponds to ORF4 in JBnB034L06 and ORF5ʹ in both HBnB148J20 and HBnB003M09, displays obvious differences among the three BAC clones (Figure 2A). Therefore, BnaA08g25920D is most likely the candidate gene for MS5. To verify this conclusion, a 3,926-bp fragment amplified from HBnB148J20 (carrying MS5a), covering the entire coding region, 1,410-bp 5ʹ upstream and 1,122-bp 3ʹ downstream sequence of BnaA08g25920D, was integrated into the binary vector pFGC5941. The construct was then introduced into the male sterile MS5bMS5b (Rs1046A) and MS5bMS5c plants. In the transformed T0 generation, 23 transgenic lines (8 for MS5bMS5b and 15 for MS5bMS5c) displayed recovered male fertility and greatly improved seed setting, while the transgene-negative plants remained unchanged (Supplemental Figure 2). Analysis of the T1 progeny from six randomly selected independent positive T0 plants showed that the fertility restoration in both MS5bMS5b and MS5bMS5c plants co-segregated with the transformation of MS5a. These results further confirmed that BnaA08g25920D is MS5. The three MS5 alleles show variations in coding and promoter sequences An 8,115-bp Mutator-like transposable element (MULE) insertion was detected in MS5b (Figure 2A), which is the only difference compared with the sequence of MS5a. However, many insertion/deletions and single nucleotide polymorphisms were

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observed in the predicted coding regions between MS5a and MS5c (Figure 2A), giving a sequence identity of 93.7% between the two MS5 alleles. To precisely determine the gene structure and allelic variations of MS5, we isolated the full-length MS5 cDNA using rapid amplification of the cDNA ends (RACE) and reverse transcription-PCR (RT-PCR). The MS5a transcript from FM195B is 1,199 bp in length, including a 10-bp 5ʹ untranslated region (UTR), a 981-bp coding sequence (CDS), and a 208-bp 3ʹ UTR (Figure 2A). The MS5c transcript from 7-5 is 1,281 bp in length, including a 51-bp 5ʹ UTR, a 1,023-bp CDS, and a 207-bp 3ʹ UTR (Figure 2A). We also attempted to isolate the full-length MS5b transcript; however, all of the resulting transcripts contained either partial sequences of the 8,115-bp MULE insertion fragment or predicted intron sequences, suggesting that the mRNA splicing of MS5b is completely altered due to the MULE insertion. Alignment of the full-length cDNA fragments and the corresponding genomic DNA sequences showed that both the MS5a and MS5c transcripts contain six exons, which is consistent with the ORF prediction for BnaA08g25920D in the reference genome (Figure 2A). Because MS5c has an extended exon 1, the coding sequence (CDS) of MS5c is 42 bp longer than that of MS5a. Though MS5a has a high CDS sequence identity (96.6%) with MS5c (Supplemental Figure 3), the presence of 33 point mutations between them leads to amino acid changes in 18 positions (Supplemental Figure 4). In addition, we found that MS5a and MS5c displayed a relatively low sequence similarity (56.1%) in the 670-bp region upstream of the transcription start site (Supplemental Figure 5). Moreover, their sequences are completely different from the upstream of the 670-bp fragment due to the specific insertion of ORF3 in the putative promoter region of MS5c (Supplemental Figure 1). MS5 encodes a novel Brassica-specific nucleus-localized protein Sequence analysis revealed that MS5 encodes a 326-amino acid protein in MS5aMS5a and a 340-amino acid protein in MS5cMS5c, both of which contain a coiled-coil domain and a Brassicaceae-specific DUF626 domain (Figure 2B and Supplemental Figure 4), as revealed by SMART analysis (http://smart.emblheidelberg.

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de). BLAST-P searches against the NCBI database (http://blast.ncbi.nlm.nih.gov), Brassica database (BRAD, http://brassicadb.org/brad/blastPage.php) and Genoscope (http://www.genoscope.cns.fr/brassicanapus/) with a reciprocal hit blast method indicated that MS5 can only identify homologs from the Brassica species (Rivera et al., 1998), including one from B. napus (BnaC08g14090D), one from B. rapa (Bra018456) and two from B. oleracea (Bol022067 and Bol022070) (Supplemental Data Set 1), and all of them carry both the predicted coiled-coil domain and the DUF626 domain. Using the primer MS5-G (Supplemental Table1), we also isolated the other two MS5 homologs from FM195A, FM195B, and 7-5, i.e., MS5-COPY2 (corresponding to BnaC08g14090D) and MS5-COPY3 that is not included in the B. napus reference genome (Chalhoub et al., 2014). We further constructed a phylogenetic tree using the above five MS5 homologs and two MS5 isoforms (Figure 2C). As expected, MS5 (BnaA08g25920D) displays the closest relationship with its B. rapa ortholog (Bra018456), while the other two B. napus MS5 paralogs, BnaC08g14090D and MS5-COPY3, seem to be the orthologs of Bol022067 and Bol022070 from B. oleracea, respectively. Together, these results indicate that MS5 is a

Brassica-specific

novel

gene

that

evolved

de

novo

after

the

Brassiceae-lineage-specific whole-genome triplication divergence of the genus Brassica from Arabidopsis, but before the speciation of B. rapa and B. oleracea. A weak nuclear localization sequence (NLS) was predicted at the N-terminus of MS5 by cNLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form. cgi; Figure 2B). To validate this prediction, we individually fused the complete MS5a and MS5c CDSs with the green fluorescent protein (GFP) gene, both driven by the 35S promoter. The previously characterized rice gene GHD7 was fused to the cyan fluorescent protein (CFP) gene as a positive control (Xue et al., 2008). MS5-GFP and the nuclear protein construct GHD7-CFP were co-transformed into Arabidopsis protoplasts. As shown in Figure 2D, the GFP and CFP signals overlapped in the nuclei of protoplasts, proving that both MS5 isoforms localize to the nucleus. The three alleles show significant differences in MS5 expression

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We examined the expression pattern of MS5 in various tissues of MS5aMS5a, MS5bMS5b (FM195A) and MS5cMS5c plants using reverse transcription quantitative PCR (RT-qPCR). To avoid potential interference from the MS5 homologs, we analyzed the CDS variations among all the homologs and designed primer sets (MS5-RT; Supplemental Table 1) specific for MS5. In each tissue, the highest accumulation of MS5 transcripts was observed in the restorer line MS5aMS5a and the lowest abundance was detected in the male sterile line MS5bMS5b, while a moderate expression level was detected in the maintainer line MS5cMS5c (Figure 2E). The peak expression of MS5 in each line was consistently detected in young buds (Figure 2E). We further investigated the MS5 level in young buds through an immunoblot using an antibody against MS5. As shown in Figure 2F, the accumulation of MS5 in MS5aMS5a was higher than that in MS5cMS5c and MS5bMS5b, with the lowest amount in MS5bMS5b. Thus, MS5 exhibits a preferential expression in reproductive organs, and has significant differences in both transcript and protein level among these three alleles. To determine the precise expression pattern of MS5 during anther development, we performed RNA in situ hybridization with anther sections of MS5aMS5a using a MS5a-derived antisense probe. Strong hybridization signals were observed in early meiocytes and tapetum (Figure 2G). However, the expression of MS5 in meiocytes decreased thereafter and was almost absent after the tetrad stage (Figure 2H to 2J). As a negative control, hybridization with the MS5a sense probe did not show obvious signals (Figure 2K). These results demonstrate that MS5 is preferentially expressed during early meiosis in B. napus. Microsporogenesis and megasporogenesis are defective in MS5bMS5b To determine how MS5 regulates plant fertility, we initially compared transverse sections of anthers at different developmental stages between MS5aMS5a and MS5bMS5b (FM195A). At early stages of anther development, primary sporogeneous cells (PSCs) developed normally in both lines (Figure 3A and 3G); then, they differentiated into microspore mother cells (MMCs), which were surrounded from

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outer to inner by epidermis, endothecium, middle layer, and tapetum (Figure 3B and 3H). However, entering meiosis, the nucleus of MMCs in MS5bMS5b was more concentrated and occupied a smaller region than those in MS5aMS5a (Figure 3B and 3H). At the end of meiosis, tetrads formed in MS5aMS5a (Figure 3C); with the degradation of callose surrounding the tetrads, individual microspores were released and further developed into mature pollen grains (Figure 3D to 3F). In contrast, tetrads were never observed inside the anther locules of MS5bMS5b at the corresponding stage (Figure 3I), indicating that the MMCs failed to complete meiosis in MS5bMS5b. These abnormal MMCs were arrested during the subsequent stages until they completely degraded during anthesis (Figure 3J to 3L). We then investigated why female fertility was severely reduced in MS5bMS5b (FM195A) plants. We first examined the ovules and found that there was no significant difference of the initial ovule number per silique between MS5aMS5a (27.9 ± 2.3; n = 30) and MS5bMS5b (28.1 ± 3.4; n = 30). When pollinated with fertile pollen, their germination and elongation were comparable in MS5aMS5a and MS5bMS5b pistils (Supplemental Figure 6A and 6C); however, unlike those in MS5aMS5a pistils (Supplemental Figure 6B), the pollen tubes failed to enter about 96.3% (n = 52) of MS5bMS5b ovules (Supplemental Figure 6D), suggesting a pollen tube targeting defect in these ovules. We further characterized the development of female gametophytes by confocal laser scanning microscopy. Typical megasporogenesis (Figure 4A to 4E) and megagametogenesis (Figure 4F to 4I) were observed in MS5aMS5a ovules; nevertheless, the process of megasporogenesis seemed to be blocked at a very early stage and no dyad or functional megaspore formed in the majority of MS5bMS5b ovules (Figure 4J to 4O), giving rise to only 16.7% (n = 107) morphologically normal female gametophytes. Therefore, the extremely low seed setting in MS5bMS5b is likely due to the arrested meiosis cell cycle of megasporocytes. We further studied the progression of female meiotic division revealed by aniline blue-stained callose in the cell plate of megasporocytes. Different callose bands were constantly observed in

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most developing megasporocytes of MS5aMS5a (Supplemental Figure 7A to 7C), whereas only smear callose was detected in the majority (83.1%, n = 118) of MS5bMS5b (FM195A) megasporocytes (Supplemental Figure 7D to 7F), indicating that most MS5bMS5b megasporocytes did not complete the first meiotic division. Taking these observations together, we proposed that both microsporogenesis and megasporogenesis are defective in MS5bMS5b. Disrupted chromosome dynamics during prophase I in MS5bMS5b meiocytes To further characterize the sterility defects in MS5bMS5b (FM195A), we investigated male meiotic chromosome spreads at different stages. In wild type (MS5aMS5a), chromosomes condensed and appeared as thin threads in the nucleus at leptotene, and several bright stained chromatin centers were visible (Figure 5A). At zygotene, the chromosome pairing occurred between homologous chromosomes (Figure 5B). At pachytene, SCs were fully formed (Figure 5C). Paired chromosomes condensed further at diplotene (Figure 5D), and became 19 distinguishable bivalents at diakinesis (Figure 5E). At metaphase I, all of these extremely condensed bivalents were arranged on the equatorial plate (Figure 5F). From anaphase I to telophase I, homologous chromosomes separated from each other and moved towards the opposite poles of the cell (Figure 5G and 5H). During meiosis II, the sister chromatids segregated equally and the dyads underwent a mitosis-like process, generating tetrads at the end of meiosis (Figure 5I). We also proved that the chromosome spreads of male meiocytes in MS5cMS5c and MS5aMS5b with normal fertility display the identical cytological patterns as MS5aMS5a (Supplemental Figure 8A to 8H). Therefore, the wild-type allotetraploid B. napus exhibited typical diploid-like meiotic behavior. In MS5bMS5b, meiotic chromosomes prepared from the young buds at leptotene were almost comparable to those in MS5aMS5a. Chromatids also showed thin thread-like structures (Figure 5J). After that, the meiotic chromosomes seemed to become diffused in the following stages. Most of them aggregated into a disordered group, while some linear threads remained at the peripheral region (Figure 5L).

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Meanwhile, nuclei became more and more compact. These chromosomes then gathered further and completely lost the typical chromosome morphology (Figure 5M). After that, chromosomes remained as a compact mass until fully degrading (Figure 5N and 5O). During the entire microsporogenesis in MS5bMS5b, bivalent formation and chromosome segregation were never detected. We examined more than 250 meiocytes at the stage after leptotene and found they all displayed the same aberrant phenotypes. In addition, we showed that the meiotic abnormalities in MS5bMS5c meiocytes strongly resembled those observed in MS5bMS5b (Supplemental Figure 8I to 8L). MS5 is required for homologous chromosome pairing To further determine the chromosome defects in MS5bMS5b, we performed fluorescence in situ hybridization (FISH) using 45S ribosomal DNA (rDNA) and 5S rDNA as probes in both MS5aMS5a and MS5bMS5b meiocytes. In MS5aMS5a, five bright hybridization 45S rDNA (n = 61, Figure 6A) and six 5S rDNA signals (n = 32, Figure 6B) with different intensities were observed in pachytene meiocytes. However, among the 24 arrested meiocytes of MS5bMS5b with quantifiable FISH signals, they all exhibited ten 45S rDNA signals. Similarly, in the investigated 20 arrested meiocytes probed with 5S rDNA, twelve FISH signals were constantly detected (Figure 6D and 6E). Therefore, the homologous chromosome pairing is severely perturbed in MS5bMS5b. Telomere bouquet formation is defective in MS5bMS5b In many species, telomeres cluster on the nuclear envelope at the beginning of zygotene forming a structure called the bouquet. It has been traditionally thought that bouquet may facilitate pairing and subsequent synapsis of the homologues, by bringing chromosome ends into close proximity. In Arabidopsis, telomeres also show a loose clustering at zygotene, which may represent a transient bouquet (Roberts et al., 2009). Because of the defective pairing in MS5bMS5b, we conducted FISH analysis with a telomere-specific probe (pAtT4) to monitor bouquet formation in MS5aMS5a

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and MS5bMS5b. In MS5aMS5a, almost all telomeres were held to a particular region, displaying the bouquet configuration at early zygotene (Figure 6C). By contrast, telomeres in MS5bMS5b were all scattered throughout the nucleus randomly (n = 59, Figure 6F). Thus, we proposed that MS5 is required for normal bouquet formation in B. napus. Accordingly, this result also supported the idea that the canonical zygotene morphology is not set up and meiocytes might be arrested before entering zygotene in MS5bMS5b. Meiotic double-strand breaks (DSBs) in MS5bMS5b The formation of a programmed set of DSBs is essential in meiosis. γH2AX, a phosphorylated form of histone H2AX, is produced in response to DSB formation and provides a sensitive indicator showing DSBs in various eukaryotes (Dickey et al., 2009). Thus, we carried out immunostaining assays using polyclonal antibodies specifically against γH2AX raised from rabbits to examine the DSBs in both MS5aMS5a and MS5bMS5b meiocytes. In MS5aMS5a, γH2AX foci appeared as numerous dot-like signals located at DAPI-stained chromatin regions from leptotene to zygotene (Figure 7A). To our surprise, in MS5bMS5b, the γH2AX localization pattern was indistinguishable from that in MS5aMS5a from leptotene through the following arrested stage (Figure 7A). Further statistical investigation revealed that there is no significant difference (P = 0.6113, unpaired t test) in the number of γH2AX foci between MS5aMS5a (289 ± 16, n = 18) and MS5bMS5b (304 ± 26, n = 15) (Figure 7B). Taken together, these data indicate that DSB formation occurs in MS5bMS5b. ASY1 and ZYP1 localization patterns during early prophase I in MS5aMS5a and MS5bMS5b The establishment of chromosome configuration at early prophase I largely depends on the installation of AEs on chromosomes. ASY1, a homolog of yeast HOP1, is an essential AE-associated protein in Arabidopsis and B. oleracea (Armstrong et al., 2002). ZYP1, an transverse filament (TF) protein, is a good marker

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to monitor the SC assembly in Arabidopsis (Higgins et al., 2005). We conducted dual immunostaining using antibodies against ASY1 and ZYP1 in MS5aMS5a and MS5bMS5b meiocytes. In MS5aMS5a, ASY1 exhibited continuous signals along unpaired chromosomal axes during leptotene, while ZYP1 showed discrete foci closely associated with the ASY1 stretches (Figure 8A). From zygotene to pachytene, accompanying SC assembly, ZYP1 signals extended rapidly and developed to form more continuous threads between homologous chromosomes. Meanwhile, filamentous ASY1 appeared to leave the chromosomes and faded to a trace after ZYP1 appeared. During pachytene, ZYP1 signals elongated on the entire length of the homologous chromosomes and only a few residual ASY1 signals were observed on chromosomes. Overlapping localization between these two proteins were rarely detected during SC installation (Figure 8A). In MS5bMS5b, ASY1 filaments were constantly detected in the meiocytes during early prophase I (Figure 8A), suggesting that the installation of axis elements occurs during early prophase I in MS5bMS5b meiocytes. Even in the arrested stage, ASY1 signals were still observed (bottom part of Figure 8A). However, no obvious ZYP1 immunostaining signals were detected in the observed meiocytes with ASY1 signals (n = 73). These results indicate that MS5 is not necessary for the proper recruiting of ASY1 but is required for central element (CE) installation during early prophase I. MS5 is required for the recruitment of SYN1 onto meiotic chromosomes Correct chromosome segregation during meiosis requires the establishment of meiosis specific sister-chromatid cohesion. Rec8 is a meiosis-specific cohesin in budding yeast that probably participates in entrapping sister chromatids (Klein et al., 1999). Homologs of yeast Rec8 have been identified in plants (Bai et al., 1999; Cai et al., 2003; Golubovskaya et al., 2006; Shao et al., 2011). Considering the severe defects in chromosome morphogenesis in MS5bMS5b meiocytes, we investigated whether the cohesin assembly is disrupted in this mutant using an Arabidopsis SYN1 antibody with ZYP1 as a control. As shown in Figure 8B, SYN1 signals formed

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consecutive linear stretches along the fully synapsed homologs at pachytene, which showed a perfect co-localization pattern with the ZYP1 filaments. Immunostaining of the SYN1 antibody was also carefully performed in MS5bMS5b meiocytes under the same condition. We lack markers specific to PMCs in MS5bMS5b; however, it is still easy to recognize the PMCs according to the DAPI staining pattern and the cytoplasm around the nucleus. For the majority of nuclei examined (73.2%, n =71), no SYN1 signal was detected during early prophase I. In other cases, weak and fuzzy SYN1 signals were occasionally observed, while no ZYP1 signals were detected in these cells (Figure 8B). Taken together, these results indicated that the proper loading of SYN1 depends on functional MS5 in B. napus. Recombination element HEI10 is absent in MS5bMS5b Since the initiation of homologous recombination normally occurs in MS5bMS5b meiocytes, we further examined whether programmed DSBs were properly repaired in those meiocytes. In Arabidopsis, HEI10 is loaded early onto numerous recombination sites during prophase I, and it remains at sites corresponding to class I crossovers until the end of recombination (Chelysheva et al., 2012). Here, we examined the distribution of HEI10 together with ASY1 by dual immunostaining. In MS5aMS5a, HEI10 appeared as intense foci and short linear signals from late leptotene to zygotene. Upon carefully investigation, we found that most HEI10 signals located on the faded ASY1 regions, which may represent the synapsed parts of the homologues (Figure 8C). The similar organization pattern between ASY1 and HEI10 has been confirmed in Arabidopsis. In contrast, although the recruitment of ASY1 seems normal, no detectable HEI10 signals were observed in all MS5bMS5b meiocytes (Figure 8C). These results suggested that MS5 is essential for homologous recombination, even it has little impact on DSB formation.

DISCUSSION Differences in fertility mediated by different MS5 alleles may depend on the expression level of MS5

16

Previous studies have indicated that the differences between three MS5 alleles lead to differences in male fertility in B. napus (Song et al., 2006; Liu et al., 2008; Lu et al., 2013). Among them, MS5a from the restorers and MS5c from the temporary maintainer are regarded as functional alleles, as these lines exhibited normal fertility. However, MS5b appears to be a loss-of-function allele due to the completely male-sterile phenotype of MS5bMS5b and MS5bMS5c. Here, we successfully isolated MS5 from the complex allopolyploid B. napus genome. Analysis of the allelic sequence differences revealed that MS5a and MS5c share highly similar CDS sequences and predicted protein sequences, but with extensive sequence polymorphisms in the promoter region. Thus, we speculate that the functional divergence between MS5a and MS5c might be caused by its different expression level induced by polymorphic promoters. In contrast, MS5b seems to be a null allele of MS5 due to its completely altered mRNA splicing caused by an 8,115-bp MULE insertion. This hypothesis was further validated by the fact that in flower buds during meiosis, MS5 has the richest transcript abundance in MS5aMS5a and a medium-low accumulation in MS5cMS5c, while it has an extremely low expression level in MS5bMS5b. Moreover, we also observed that MS5 had a greatly reduced transcript level in MS5bMS5b and MS5bMS5c compared with MS5cMS5c, but maintained about two-fold higher abundance in MS5aMS5b than in MS5cMS5c (Supplemental Figure 9). More importantly, immunoblot analysis revealed that the accumulation of MS5 displayed a highly similar tendency between MS5aMS5a, MS5bMS5b and MS5cMS5c. Taking our results together, we believe that the fertility transition regulated by different alleles of MS5 depends on their significant differences in expression, which eventual leads to the haploinsufficiency of the MS5 protein in MS5bMS5c. Haploinsufficiency refers to a mutant phenotype exhibited in heterozygotes. Due to only half the normal amount of gene product being transcribed, it is not sufficient to maintain the normal phenotype in a heterozygote (Veitia, 2002; Meinke, 2013). Thus, the quantity of functional MS5c might not meet the threshold for normal male meiosis in MS5bMS5c, while the quantity of functional MS5a far exceeds its minimal

17

requirement. A recent example of similar dose-limiting effect in meiosis is the HOP2/MND1 complex, which promotes interhomolog DNA repair by stimulating DMC1 activity in a dosage-sensitive manner (Uanschou et al., 2013). Interestingly, in this study, we also observed that male meiosis is completely disrupted in MS5bMS5c, while the female meiosis largely proceeds normally. Even in MS5bMS5b, there are still about 16.7% morphologically-normal female gametophytes generated. Thus, female meiosis seems less sensitive to the haploinsufficiency of MS5 compared with male meiosis. The coiled-coil domain of MS5 might be crucial for meiosis Sequence analysis showed that MS5 shares very low similarity with any proteins outside of Brassica species. Two putative conserved structures reside in MS5, i.e., the DUF626 domain and a coiled-coil domain. The coiled-coil domain may facilitate protein-protein interactions by forming a coiled-coil dimer (Lupas, 1996). Indeed, heterodimers of SMC family proteins carrying the coiled-coil domain function directly in sister chromatid cohesion or chromosome condensation during both mitosis and meiosis (Nasmyth and Haering, 2005). Proteins with a putative coiled-coil domain, such as SWI1 (Mercier et al., 2001), AM1 (Che et al., 2011), PAIR1 (Nonomura et al., 2004), and PAIR3 (Yuan et al., 2009), are also required for meiosis initiation, homologous chromosome pairing, synapsis, and recombination. In addition, all transverse filament proteins of SCs in different organisms have significantly similar structures, with a coiled-coil domain in the central region, even they share very low similarity at the amino acid level. Thus, proteins with the coiled-coil domain display functional diversity during meiosis. Since MS5 is a novel Brassica-specific protein, it remains a challenge to deduce its detailed roles in meiosis according to the conserved coiled-coil domain. MS5 probably functions in establishing meiosis-specific chromosome structure during early prophase I In the present study, MS5b MS5b MMCs presented normal meiotic chromosome

18

configuration only at leptotene, but lost distinct chromosome morphology as well as cytokinesis during the following stages. This mirrors the observed phenotype of leptotene arrest in maize am1-praI allele and the rice am1 mutant (Golubovskaya et al., 1993; Che et al., 2011), indicating the indispensable role of MS5 in meiosis progression. Moreover, meiosis-specific SC assembly and homologous recombination, indicated by ZYP1 and HEI10 signals, are both lost in MS5bMS5b MMCs, raising the possibility that MS5 functions in chromosome configuration and early meiotic events. In budding yeast, Red1 has been described as a meiotic chromosome core protein (Smith and Roeder, 1997). Its homologs have recently been found in rice (PAIR3), Arabidopsis (ASY3) and maize (DSY2) (Wang et al., 2011; Ferdous et al., 2012; Lee et al., 2015). All of them are required for the proper localization of the HORMA domain protein Hop1/PAIR2/ASY1, an axis-associated protein (Caryl et al., 2000; Armstrong et al., 2002; Nonomura et al., 2006; Shao et al., 2011). In MS5bMS5b MMCs, almost normal ASY1 signals exist in early prophase I, indicating that the loading of ASY1 might have occurred by leptotene. During prophase I, cohesins are involved in AE formation and SC assembly from yeast to plant. Recently studies in Arabidopsis found that the localization of ASY1 is dependent on both ASY3 and SYN1, but the relationship is not reciprocal (Ferdous et al., 2012). The aberrant localization of ASY1 and PAIR2 is also observed in maize afd1 and rice rec8 (Golubovskaya et al., 2006; Shao et al., 2011). However, the loss of MS5 has a profound defect on the recruitment of SYN1 onto meiotic chromosomes, but has no detectable influence on ASY1 loading. Previous immunogold assays in B. oleracea showed that during meiotic interphase, ASY1 is distributed over the diffuse chromatin prior to the appearance of AEs (Armstrong et al., 2002). In Arabidopsis, chromosome axis formation occurs in conjunction with the association of ASY1, and axis morphogenesis is independent of ASY1 (Sanchez-Moran et al., 2007). Since B. oleracea is one diploid ancestor species of B. napus, we propose that the association of ASY1 with axes possibly occurs earlier than the installation of SYN1 at the early stage of meiosis, and to some degree the two processes might be independent of each

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other. In addition, the tapetum is important in the development of MMCs in plants. Our in-situ RNA analyses revealed that MS5 is expressed both in meiocytes and tapetum. This expression pattern is similar to the rice DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1), and mutation in DTM1 leads to the arrest of MMCs in early prophase I (Yi et al., 2012). Taken together, our studies implied that MS5 is necessary for proper chromosome organization during early prophase I, possibly acting directly in meiosis, or indirectly, like DTM1 in rice.

METHODS Plant materials and growth conditions Brassica napus Rs1046A (MS5bMS5b) is a genetic male sterile (GMS) line transferred from a spontaneous male-sterile mutation, Yi3A (Zhou and Bai, 1994; Lu et al., 2013). By using Rs1046A as the donor and a GMS restorer (DH195) as the recipient, we generated another GMS line FM195A (MS5bMS5b) and its male-fertile nearly isogenic lines (NILs), FM195H (MS5aMS5b) and FM195B (MS5aMS5a). The wild-type double-haploid accession 7-5 (MS5cMS5c) is a temporary maintainer line of Rs1046A and FM195A, since the male sterility of the resulting F1 plants (MS5bMS5c) is unable to be fully maintained by backcrossing with 7-5. All the B. napus lines were grown in the experimental farm of Huazhong Agricultural University during the normal growing season in Wuhan, China. Arabidopsis thaliana plants (ecotype Columbia-0) used for protein localization were grown in a growth chamber at 22 ± 2°C under a 16 h light/8 h dark photoperiod. BAC clone library, screening, and sequencing A B. napus BAC library (HBnB) was constructed by using the genomic DNA of F1 plants from a cross between Rs1046A (MS5bMS5b) and DH195B (transferred from DH195, MS5aMS5a) (Deng et al., 2016). BAC clone plasmid DNA was prepared with a Macherey-Nagel NucleoBond Xtra Midi Kit (MN, Germany). The target BAC

20

clones were identified through a two-stage PCR screening method using three pairs of primers flanking or co-segregating with MS5 (BE10, G3-2, and WKU-2; Supplemental Table 1). G3-2 is a dominant marker linked with MS5a designed according the sequence of BnaA08g25920D. Dominant marker WKU-2 linked with MS5b was developed according to the sequence of BnaA08g25920D. All the markers were validated in the F2 or BC1 segregation populations. Complementation test For the functional complementation test, a ~3.9-kb genomic DNA fragment covering ORF5’ was amplified from the MS5a-containing BAC clone HBnB148J20 using primer pair G5-F/R (Supplemental Table 1), with Phusion High-Fidelity DNA polymerase (Thermo Scientific). The fragment was cloned into the binary vectors pFGC5941 and the appropriate construct was introduced into Agrobacterium tumefaciens GV3101 host cells. The complementation construct was introduced into the recipients FM195A (MS5bMS5b), as well as the male-sterile F1 plants (MS5bMS5c) produced

from

a

cross

between

Rs1046A

and

7-5,

via

Agrobacterium

tumefaciens-mediated transformation. Homozygous FM195A plants were selected by genotyping of the seedlings (at the cotyledon stage) derived from FM195H (MS5aMS5b). Positive transgenic events were selected by PCR analysis with two insertion-specific

primer

pairs

(MS5a-V-L1/MS5a-G-R1;

G3-2L/G3-2R)

(Supplemental Table 1). The individual fertility of transgenic plants was visually investigated and further confirmed by microscopic examination of pollen stained with 0.5% acetocarmine solution. Number of seeds per silique was measured as a mean from ten well-developed siliques sampled from the middle region of a primary branch. Anther cross sectioning and callose detection Flower buds from FM195A and FM195B were collected for cross section preparation as described by (Wan et al., 2010b). The sections were photographed under an Axio Scope A1 microscope (Carl Zeiss). To detect the callose deposition in

21

pollen germination and pollen tube growth, the stigmas from flowers one day before anthesis were artificially pollinated with mature pollen grains. Gynoecia collected at 2, 4, 24, 48 h after pollination were then fixed in formalin-acetic acid-alcohol (FAA) solution (5% formaldehyde, 5% glacial acetic acid, and 63% ethanol) for 24 h. After rinsing in water, the samples were softened with 6 M NaOH for 12 h at room temperature, washed twice in distilled water, and incubated for 24 h in 0.1% aniline blue solution containing 0.14 M K2HPO4 (pH 8.2) at room temperature. The stained samples were mounted in 30% glycerol and observed under an Axio Scope A1 fluorescence microscope (Carl Zeiss) with a UV filter. Observation for callose deposition in the meiotic megasporocytes and megaspores was as described (Li et al., 2015). Confocal microscopy of ovule development To examine ovule development in FM195A and FM195B, ovules at different developmental stages were dissected and prepared for microscopy according to the protocol described before (Zeng et al., 2009), and were imaged on a confocal laser scanning microscope (TCS SP2, Leica) with an excitation wavelength of 559 nm and detection wavelengths of 603-637 nm. RACE and RT-qPCR analysis Total RNA was isolated from various tissues and flower buds during meiosis from different accessions or F1 plants, using an RNeasy Plant Mini Kit (QIAGEN). The cDNA was reverse transcribed using the MMLV system (Promega, http://www.promega.com.cn/) with 0.5 µg of oligo (dT)18 and 2 µg of total RNA in a 25 µL reaction. To obtain full-length transcripts of MS5, RT-PCR, 5’-RACE and 3’-RACE were performed with a SMART RACE cDNA amplification kit (Clontech) according to the manufacturer’s instructions. CDS of MS5 and its homeologues were amplified with a conserved primer pair MS5-G using cDNA of FM195B, FM195A and 7-5 as the template. For 5’-RACE, nested primers (RACE5-ABCGSP2 and RACE5-ABCNGSP2) specific for MS5a and MS5c were designed according to the

22

SNP differences between MS5 and its two homologues. The 3’ RACE was conducted with conserved primers (RACE3-3GSP1 and RACE3-3NGSP1), and the isolated sequences were then grouped to different copies according to their specific SNPs. The 50ng/µl diluted cDNA was used as templates for subsequent real-time PCR of MS5 in three replications using the gene-specific primer pair MS5-RT-F/R with the CFX96 Real-time system (Bio-Rad). The expression level of MS5 was calculated after normalization to that of ACTIN7 as an internal control using CFX Manager software according to the 2-ΔΔCt method (Livak and Schmittgen, 2001). All primers are listed in Supplemental Table 1. Database searches and sequence analysis BLAST searches were conducted at the NCBI, Genoscope and Brassica database (BRAD). The phylogenetic tree of MS5 was constructed using MEGA6 (http://www.megasoftware.net/) based on the neighbor-joining method with parameters of Poisson correction model, pairwise deletion, and bootstrap (1000 replicates; random seed) (Tamura et al., 2013). The nucleotide and amino acid sequences were analyzed using Geneious 6.1.8. Antibodies To generate the antibody against MS5, the entire coding region (978 bp) of MS5a cDNA was amplified using primers MS51F/MS51R (see Supplemental Table 1). The PCR product was inserted into the vector pGEX-6P-1 (Amersham) using enzyme digestion and ligation. The target plasmid was then transformed into Escherichia coli strain BL21 (DE3). The fusion peptides were accumulated predominantly in the form of inclusion bodies. The target protein was used to immune the mouse and generate the antibody. Primary antibodies against ASY1 (rabbit), SYN1 (rabbit) and ZYP1 (mouse) were used for the immunofluorescence assays (Wang et al., 2012b). The primary antibodies against γH2AX (rabbit) and HEI10 (mouse) were obtained in our previous studies (Wang et al., 2012a; Miao et al., 2013). The secondary antibodies were goat

23

anti-mouse with FITC conjugate and goat anti-rabbit with TRITC conjugate (Southern Biotech). Protein extraction and immunoblot analysis Fresh flower buds during early meiosis (1-2mm) were ground into powder in liquid nitrogen, and immediately incubated in 5% SDS solution for total protein extraction. Protein samples were separated by 12% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) type membrane (Millipore) using a Bio-Rad mini transfer cell. Immunoblots were incubated with anti-MS5 antiserum diluted 1:5,000 followed by a Goat Anti-Mouse IgG antibody conjugated to horseradish peroxidase (Abmart) diluted 1:5,000. An anti-mouse actin antibody for plants (Abmart) was used as a positive control. Signals were visualized by a Keygen ECL system and scanned with a Clinx ChemiScope chemiluminescence imaging system (Kaiji). RNA in situ hybridization Preparation of anther cross sections, RNA in situ hybridization, and immunologic detection were performed as described (Wan et al., 2010b). A 486-bp cDNA fragment from MS5a was amplified using the primer pair ABCRT-Xba1-L/R (Supplemental Table 1) and inserted into the PSPT18 vector (Roche). The antisense and sense RNA probes were then produced from the T7 and SP6 promoters with polymerase using digoxigenin RNA labeling reagents (Roche). Protein subcellular localization The full-length CDSs of MS5a and MS5c without the termination codon were individually amplified with the primer pairs MS5a-pM999-F/R and MS5c-pM999-F/R (Supplemental Table 1) and cloned into the pM999-GFP vector. The rice nuclear protein GHD7 fused with CFP was used as a nuclear marker (Xue et al., 2008). The fusion constructs were introduced into Arabidopsis mesophyll protoplasts by PEG/calcium-mediated transformation (Yoo et al., 2007). Fluorescence in the transformed protoplasts was imaged under a confocal laser scanning microscope (TCS SP2, Leica) with standard excitation and detection wavelengths.

24

Meiotic chromosome preparation Young inflorescences of B. napus at the proper stages were harvested and fixed in Carnoy’s solution (ethanol:glacial acetic acid, 3:1) for at least 48 h at room temperature. A flower bud in an inflorescence was dissected, and anthers from the individual bud were obtained. The anthers were quickly and completely squashed with a dissecting needle on a glass slide, and the MMCs in anthers were released into acetocarmine solution. The sample was covered with a 22 × 22 mm coverslip. MMCs were detected under a phase contrast microscope to assess the meiotic stages. Slides with MMCs undergoing meiosis were chosen, and acetocarmine in these slides was faded with 45% glacial acetic. For DAPI-staining, each slide was frozen in liquid nitrogen and the coverslip was removed with a razor blade. An alcohol series comprising absolute ethanol and sterile deionized water at 70%, 90%, and 100% ethanol (5 min each wash) was used for slide dehydration. Chromosomes were counterstained with DAPI in antifade mounting medium (Vector Laboratories). Original images were captured under a ZEISS imager A2 fluorescence microscope equipped with a 100× lens and a high-resolution CCD camera. Fluorescence in situ hybridization (FISH) FISH analysis was performed as previously described with slight modifications (Zhang et al., 2005). The 45S rDNA, 5S rDNA and pAtT4 were labeled with digoxigenin-11-dUTP

and

used

as

FISH

probes.

FITC-conjugated

sheep

anti-digoxigenin (Roche) was used to detect the probes. Immunofluorescence A modified immunofluorescence assay was performed based on the previously described method (Cheng, 2013). Fresh inflorescences of B. napus were fixed in 4% (w/v) paraformaldehyde for 30-50 min at 4°C and washed three times in 1× PBS on ice (pH 8.0, 5 min per wash). Anthers at the desired meiotic stage were dissected and squashed on a slide with 1× PBS solution. Immunostaining was performed at 4°C

25

overnight, and the slides were incubated with the desired primary antibodies diluted at 1:500 in 1× PBS. After three rounds of 5-min washing in 1× PBS, the slides were incubated with 1,000-fold-diluted fluorophore-coupled secondary antibodies (goat anti-mouse with FITC conjugate, goat anti-rabbit with TRITC conjugate) at 37°C for 2 h and washed three times (5 min per wash). The slides were then air-dried and the chromosome spreads were counterstained with DAPI. Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: MS5c, KX223881; MS5a, KX223882; MS5-COPY2, KX231797; MS5-COPY3, KX231798; Bol022067, KX231800; Bol022070, KX231801; Bra018456, KX231799; ACTIN7, NM_001316010; SYN1, AT5G05490; ASY1, AT1G67370; ZYP1, AT1G22260; HEI10, AFJ04809; GHD7, EU286800.

SUPPLEMENTAL DATA Supplemental Figure 1. Map-based cloning of MS5. Supplemental Figure 2. Complementation test of MS5. Supplemental Figure 3. Comparison of the coding sequences of MS5a and MS5c. Supplemental Figure 4. Comparison of the amino acid sequences of MS5a and MS5c Supplemental Figure 5. Sequence alignment of the MS5a and MS5c promoter regions. Supplemental Figure 6. Fluorescence micrographs of pollen tube germination and growth in MS5aMS5a and MS5bMS5b. Supplemental Figure 7. Meiotic division of megasporogenesis is blocked in MS5bMS5b. Supplemental Figure 8. Meiotic chromosome dynamics of different meiotic stages in MS5c MS5c, MS5a MS5b and MS5b MS5c MMCs. Supplemental Figure 9. Expressional levels of MS5 in plants with different allele



26

combinations. Supplemental Table 1. Summary of the primers used in this study. Supplemental Data Set 1. Text file of the alignment corresponding to the phylogenetic tree in Figure 2C.

ACKNOWLEDGEMENTS We are grateful to Dr. Yingxiang Wang in Fudan University for providing Arabidopsis antibodies. We also thank Dr. Xianhong Ge in Huazhong Agriculture University for valuable comments and discussion. This work was supported by grants from the National Natural Science Foundation of China (31230038 and 31500255), the Ministry of Sciences and Technology of China (2011AA10A104), and the Project 2013PY040 supported by the Fundamental Research Funds for the Central Universities.

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Figure Legends Figure 1. Fertility Performances of Plants with Different MS5 Alleles. From top to bottom, petal-removed flowers (A) to (E), enlarged anthers (F) to (J), and mature siliques under artificial pollination (K) to (O), are shown in each row, respectively. (A), (F) and (K) MS5aMS5a. (B), (G) and (L) MS5cMS5c. (C), (H) and (M) MS5bMS5b (FM195A). (D), (I) and (N) MS5bMS5b (Rs1046A). (E), (J) and (O) MS5bMS5c. Bars, 2 mm in (A) to (E), 500 µm in (F) to (J), and 1 cm in (K) to (O). Figure 2. Molecular Characterization of MS5. (A) Genomic structure of MS5a, MS5b, and MS5c. The six red arrowed boxes indicate numbered exons. The 5’ UTR and 3’ UTR are separately shown by green arrowed boxes. An 8,115-bp Mutator-like transposable element (MULE) insertion in the 520-bp of MS5a gives rise to MS5b. The spaces on the thick black lines indicate the sequence variations between MS5a and MS5c. (B) Protein structure of MS5a and MS5c. (C) Phylogenetic tree of MS5 homologs. MS5a, MS5c, and the five MS5 homologs were selected to establish a bootstrap N-J phylogenetic tree, with bootstrap values (%) from 1,000 replicates. Black dots show the positions of MS5a and MS5c. The lengths of the branches refer to the amino acid variation rates. (D) Subcellular localization of full-length MS5a and MS5c fused with green fluorescent protein (GFP); the nuclear protein GHD7-fused cyan fluorescent protein (CFP) was used as a nuclear marker. Bar = 10 µm. (E) RT-qPCR analysis of MS5 in MS5aMS5a, MS5bMS5b (FM95A), and MS5cMS5c respectively. 1, 4, and 7, flower buds during meiosis; 2, 5, and 8, leaves; 3, 6, and 9, stems. Expression values are means ± s. d, based on three biological replicates. (F) Immunoblot detection of MS5 using anti-MS5 bodies in flower buds of MS5aMS5a, MS5bMS5b (FM95A), and MS5cMS5c at meiosis stage.

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(G) to (K) RNA in situ hybridization of MS5a at a range of developmental stages in MS5aMS5a anthers. MMC, microspore mother cell; T, tapetum; Tds, tetrads; Msp, microspore. Bars = 50 µm (G) and (H) Early and late meiosis stages, respectively. (I) Late tetrad stage. (J) Microspore stage. (K) No signal was detected at the microspore stage with MS5a sense probe. Figure 3. Transverse Sections of Anthers in MS5aMS5a and MS5bMS5b. (A-F) Anthers of MS5aMS5a, different pollen development stages are shown, including the PSC stage (A), MMC stage (B), tetrad stage (C), young microspore stage (D), late microspore stage (E), and mature pollen stage (F). (G-L) Anthers of MS5bMS5b (FM195A), PSCs differentiate normally (F) and proceed into the MMC stage with abnormal nuclei (H), while none subsequent meiotic stages were detected (I-L). PSC, primary sporogenous cell; E, epidermis; En, endothecium; ML, middle layer; T, tapetum; MMC, microspore mother cell; Tds, tetrads; Msp, microspore; PG, pollen grain; DM, degenerating meiocytes. Bars = 50 µm. Figure 4. Confocal Laser Scanning Microscopy Images of Ovules in MS5aMS5a and MS5bMS5b. (A) to (I) Normal ovule development in MS5aMS5a. (A) and (B) Early and late megasporocyte stage. (C) Megasporocyte meiosis stage; (D) Early tetrad stage. (E) Tetrad stage. (F) Mono-nucleate embryo sac stage. (G) Two-nucleate embryo sac stage. (H) Four-nucleate embryo sac stage. (I) Mature embryo sac. (J) to (O) Defective ovule development in MS5bMS5b. Megasporocyte differentiates normally (J) and further progresses into the meiosis stage (K). Normal tetrads (L) and functional megaspores were rarely observed. In the subsequent stages (M) and (N), none typical embryo sacs were observed. II, internal integument; OI, outer integument; MMC, megaspore mother cell; Te, tetrad; FM, functional megaspore; DM, degenerating megaspore; CC, central cell; AMMC, abnormal megaspore mother cell; AES, abnormal embryo sac. Bar = 100 µm. Figure 5. Meiotic Chromosomes Stained with DAPI in MS5aMS5a and MS5bMS5b MMCs. (A) to (I) MS5aMS5a. (A) Leptotene; (B) Zygotene. Homologous chromosomes pair and begin to form SCs.

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(C) Pachytene. Chromosomes condense further and SCs assemble along the full-length of chromosomes. (D) Diplotene. (E) Diakinesis. Paired homologous chromosomes are attached to each other only at the chiasmata, the physical connections corresponding to crossovers. (F) Metaphase I. The extremely condensed bivalents line up on the equatorial plate; (G) Anaphase I. Homologous chromosomes segregate equally; (H) Telophase I; (I) Tetrad. (J) to (O) MS5bMS5b. (J) Leptotene. Chromosomes are as thread-like as in MS5aMS5a. (K) Meiocytes start to show arrested phenotype and chromosomes become diffused. (L) and (M) Chromosomes become more diffused and nuclei become more compact. (N) and (O) The final arrested MMCs. Chromosomes remain together as a diffused mass. Neither meiosis I nor meiosis II is completed in these MMCs, showing meiosis being completely arrested. Bars = 5 µm. Figure 6. FISH Analysis in MS5aMS5a and MS5bMS5b MMCs Probed with 45S rDNA, 5S rDNA and Telomere-specific Clone pAtT4. (A) to (C) Meiocytes in MS5aMS5a. Five FISH signals for 45S rDNA (A) and six for 5S rDNA (B) with different intensity are detected in the pachytene nuclei. pAtT4 signals are clustered, showing telomeres forming a transient bouquet at early zygotene (C). (D) to (F) Meiocytes in MS5bMS5b. Ten 45S rDNA (D) and twelve 5S rDNA signals (E) are shown in the arrested nuclei, indicating that homologous chromosomes without pairing in MS5bMS5b meiocytes. pAtT4 signals are scattered, indicating a failure in telomere bouquet formation (F). Chromosomes were stained with DAPI and are pseudo colored in red. Bars = 5 µm. Figure 7. Immunolocalization of the γH2AX in MS5aMS5a and MS5bMS5b MMCs. (A) γH2AX staining appears as punctate foci in both MS5aMS5a and MS5bMS5b meiocytes. (B) The numbers of γH2AX foci are quantified in MS5aMS5a and MS5bMS5b (P = 0.6113, unpaired t test). ns, not statistically different. DAPI staining chromosomes in blue. γH2AX signals in red. Bars = 5 µm. Figure 8. Immunodetection of Meiotic Elements in MS5aMS5a and MS5bMS5b MMCs. (A) In MS5aMS5a, ASY1 (Red) shows long thread at late leptotene, and ZYP1 (Green) is detected as punctate foci at this stage. ASY1 gradually releases from the synapsis regions of chromosomes from zygotene to pachytene. ZYP1 elongates along the paired homologous chromosomes rapidly and forms continuous linear signals. At pachytene, a few residual ASY1 signals are left in the nucleus, while SC assembly is

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completed. In MS5bMS5b, ASY1 signals undergo nearly normal localization progression from leptotene (the upper part) through the post arrested stage (the bottom part). However, no obvious ZYP1 signals were detected. (B) In MS5aMS5a, SYN1 (Red) co-localizes with ZYP1 (Green) along the whole chromosomes at pachytene. In most cases, no SYN1 signal was detected in MS5bMS5b meiocytes. In the left cases, some faint and diffused SYN1 signals were observed, however, no ZYP1 signals were observed even in the nuclei with faint SYN1 foci. Chromosomes are in blue (DAPI-stained). (C) In MS5aMS5a, HEI10 (Green) displays as foci and short linear signals in late leptotene and zygotene nuclei, while no detectable HEI10 signal was observed in MS5bMS5b. Bars = 5 µm.



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Figure 1. Fertility performances of plants with different MS5 alleles. From top to bottom, petal-removed flowers (A) to (E), enlarged anthers (F) to (J), and mature siliques under artificial pollination (K) to (O), are shown in each row, respectively. (A), (F) and (K) MS5aMS5a. (B), (G) and (L) MS5cMS5c. (C), (H) and (M) MS5bMS5b (FM195A). (D), (I) and (N) MS5bMS5b (Rs1046A). (E), (J) and (O) MS5bMS5c. Scale bars, 2 mm in (A) to (E), 500 µm in (F) to (J), and 1 cm in (K) to (O).

Figure 2. Molecular characterization of MS5. (A) Genomic structure of MS5a, MS5b, and MS5c. The six red arrowed boxes indicate numbered exons. The 5’ UTR and 3’ UTR are separately shown by green arrowed boxes. An 8,115-bp Mutatorlike transposable element (MULE) insertion in the 520-bp of MS5a gives rise to MS5b. The spaces on the thick black lines indicate the sequence variations between MS5a and MS5c. (B) Protein structure of MS5a and MS5c. (C) Phylogenetic tree of MS5 homologs. MS5a, MS5c, and the five MS5 homologs were selected to establish a bootstrap N-J phylogenetic tree, with bootstrap values (%) from 1,000 replicates. Black dots show the positions of MS5a and MS5c. The lengths of the branches refer to the amino acid variation rates. (D) Subcellular localization of full-length MS5a and MS5c fused with green fluorescent protein (GFP); the nuclear protein GHD7-fused cyan fluorescent protein (CFP) was used as a nuclear marker. Scale bars, 10 µm. (E) qRT-PCR analysis of MS5 in MS5aMS5a, MS5bMS5b (FM95A), and MS5cMS5c respectively. 1, 4, and 7, flower buds during meiosis; 2, 5, and 8, leaves; 3, 6, and 9, stems. Expression values are means ± s. d, based on three biological replicates. (F) Immunoblot detection of MS5 using anti-MS5 bodies in flower buds of MS5aMS5a, MS5bMS5b (FM95A), and MS5cMS5c at meiosis stage. (G) to (K) RNA in situ hybridization of MS5a at a range of developmental stages in MS5aMS5a anthers. (G) and (H) Early and late meiosis stages, respectively; (I) Late tetrad stage; (J) Microspore stage. No signal was detected at the microspore stage with MS5a sense probe (K). MMC, microspore mother cell; T, tapetum; Tds, tetrads; Msp, microspore. Scale bars, 50 µm.

Figure 3. Microsporogenesis is arrested in MS5bMS5b. Transverse sections of anthers in both MS5aMS5a and MS5bMS5b. (A-F) Anthers of MS5aMS5a, different pollen development stages are shown, including the PSC stage (A), MMC stage (B), tetrad stage (C), young microspore stage (D), late microspore stage (E), and mature pollen stage (F). (G-L) Anthers of MS5bMS5b (FM195A), PSCs differentiate normally (F) and proceed into the MMC stage with abnormal nuclei (H), while none subsequent meiotic stages were detected (I-L). PSC, primary sporogenous cell; E, epidermis; En, endothecium; ML, middle layer; T, tapetum; MMC, microspore mother cell; Tds, tetrads; Msp, microspore; PG, pollen grain; DM, degenerating meiocytes. Scale bars, 50 µm.

Figure 4. Megasporogenesis and megagametogenesis are defective in MS5bMS5b. Confocal laser scanning microscopy images of ovules in both MS5aMS5a and MS5bMS5b. (A) to (I) Normal ovule development in MS5aMS5a. Successive developmental stages of the ovule were observed. (A) and (B) Early and late megasporocyte stage; (C) Megasporocyte meiosis stage; (D) Early tetrad stage; (E) Tetrad stage; (F) Mono-nucleate embryo sac stage; (G) Two-nucleate embryo sac stage; (H) Four-nucleate embryo sac stage; (I) Mature embryo sac. (J) to (O) Defective ovule development in MS5bMS5b. Megasporocyte differentiates normally (J) and further progresses into the meiosis stage (K). Normal tetrads (L) and functional megaspores were rarely observed. In the subsequent stages (M) and (N), none typical embryo sacs were observed. Ovules and embryo sacs in MS5bMS5b are much smaller than those in MS5aMS5a at the corresponding stages. II, internal integument; OI, outer integument; MMC, megaspore mother cell; Te, tetrad; FM, functional megaspore; DM, degenerating megaspore; CC, central cell; AMMC, abnormal megaspore mother cell; AES, abnormal embryo sac. Scale bar, 100 µm.

Figure 5. Meiotic chromosomes stained with DAPI in both MS5aMS5a and MS5bMS5b MMCs. (A) to (I) MS5aMS5a. (A) Leptotene; (B) Zygotene. Homologous chromosomes pair and begin to form SCs; (C) Pachytene. Chromosomes condense further and SCs assemble along the full-length of chromosomes; (D) Diplotene; (E) Diakinesis. Paired homologous chromosomes are attached to each other only at the chiasmata, the physical connections corresponding to crossovers; (F) Metaphase I. The extremely condensed bivalents line up on the equatorial plate; (G) Anaphase I. Homologous chromosomes segregate equally; (H) Telophase I; (I) Tetrad. (J) to (O) MS5bMS5b. (J) Leptotene. Chromosomes are as thread-like as in MS5aMS5a; (K) Meiocytes start to show arrested phenotype and chromosomes become diffused; (L) and (M) Chromosomes become more diffused and nuclei become more compact; (N) and (O) The final arrested MMCs. Chromosomes remain together as a diffused mass. Neither meiosis I nor meiosis II is completed in these MMCs, showing meiosis being completely arrested. Scale bars, 5 µm

Figure 6. FISH analysis in both MS5aMS5a and MS5bMS5b MMCs. Meiotic chromosomes probed with 45S rDNA (Green), 5S rDNA (Green) and telomerespecific clone pAtT4 (Green), respectively. Chromosomes were stained with DAPI and are pseudo colored in red. (A) to (C) Meiocytes in MS5aMS5a. Five FISH signals for 45S rDNA (A) and six for 5S rDNA (B) with different intensity are detected in the pachytene nuclei. (C) pAtT4 signals are clustered, showing telomeres forming a transient bouquet at early zygotene. (D) to (F) Meiocytes in MS5bMS5b. Ten 45S rDNA (D) and twelve 5S rDNA signals (E) are shown in the arrested nuclei, indicating that homologous chromosomes without pairing in MS5bMS5b meiocytes. (F) pAtT4 signals are scattered, indicating a failure in telomere bouquet formation. Scale bars, 5 µm

Figure 7. Immunolocalization of the γH2AX in both MS5aMS5a and MS5bMS5b MMCs. DAPI staining (Blue) indicating the meiotic chromosomes. γH2AX signals (Red) showing with DSB formation. (A) γH2AX staining appears as punctate foci in both MS5aMS5a and MS5bMS5b meiocytes; (B) The numbers of γH2AX foci are quantified in MS5aMS5a and MS5bMS5b (P = 0.6113, unpaired t test). ns, not statistically different. Scale bars, 5 µm.

Figure 8. Immunodetection of meiotic elements in both MS5aMS5a and MS5bMS5b MMCs. (A) In MS5aMS5a, ASY1 (Red) shows long thread at late leptotene, and ZYP1 (Green) is detected as punctate foci at this stage. ASY1 gradually releases from the synapsis regions of chromosomes from zygotene to pachytene. ZYP1 elongates along the paired homologous chromosomes rapidly and forms continuous linear signals. At pachytene, a few residual ASY1 signals are left in nucleus, while entire SCs assembly is fulfilled. In MS5bMS5b, ASY1 signals undergo nearly normal localization progression from leptotene (the upper part) through the post arrested stage (the bottom part). However, no obvious ZYP1 signals were detected. (B) In MS5aMS5a, SYN1 (Red) co-localizes with ZYP1 (Green) along the whole chromosomes at pachytene. In most cases, no SYN1 signal was detected in MS5bMS5b meiocytes. In the left cases, some faint and diffused SYN1 signals were observed, however, no ZYP1 signal were observed even in the nuclei with faint SYN1 foci. Chromosomes are in blue (DAPI-stained). (C) In MS5aMS5a, HEI10 (Green) displays as foci and short linear signals in late leptotene and zygotene nuclei, while no detectable HEI10 signal was observed in MS5bMS5b. Scale bars, 5 µm.

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MS5 mediates early meiotic progression and its natural variants may have applications for hybrid production in Brassica napus Qiang Xin, Yi Shen, Xi Li, Wei Lu, Xiang Wang, Xue Han, Faming Dong, Lili Wan, Guangsheng Yang, Dengfeng Hong and Zhukuan Cheng Plant Cell; originally published online May 18, 2016; DOI 10.1105/tpc.15.01018 This information is current as of May 20, 2016 Supplemental Data

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