The Role of OsMSH5 in Crossover Formation during Rice Meiosis

Molecular Plant  •  Volume 6  •  Number 3  •  Pages 729–742  •  May 2013

RESEARCH ARTICLE

The Role of OsMSH5 in Crossover Formation during Rice Meiosis Qiong Luoa,2, Ding Tangb,2, Mo Wangb, Weixiong Luoa, Lei Zhangb, Baoxiang Qinb, Yi Shenb, Kejian Wangb, Yafei Lib and Zhukuan Chengb,1 a College of Plant Protection, Yunnan Agricultural University, Kunming 650201, 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

ABSTRACT  MSH5, a meiosis-specific member of the MutS-homolog family, is required for normal level of recombination in budding yeast, mice, Caenorhabditis elegans, and Arabidopsis. Here, we report the identification and characterization of its rice homolog, OsMSH5, and demonstrate its function in rice meiosis. Five independent Osmsh5 mutants exhibited normal vegetative growth and severe sterility. The synaptonemal complex is well installed in Osmsh5, while the chiasma frequency is greatly reduced to approximately 10% of that observed in the wild-type, leading to the homologous nondisjunction and complete sterile phenotype. OsMSH5 is predominantly expressed in panicles. Immunofluorescence studies indicate that OsMSH5 chromosomal localization is limited to the early meiotic prophase I. OsMSH5 can be loaded onto meiotic chromosomes in Oszip4, Osmer3, and hei10. However, those ZMM proteins cannot be localized normally in the absence of OsMSH5. Furthermore, the residual chiasmata were shown to be the least frequent among the zmm mutants, including Osmer3, Oszip4, hei10, and Osmsh5. Taken together, we propose that OsMSH5 functions upstream of OsZIP4, OsMER3, and HEI10 in class I crossover formation. Key words: OsMSH5; crossover; meiosis; rice.

Introduction Meiosis is a specialized type of cell division in gamete genesis. During this process, a single round of DNA replication is followed by two rounds of chromosome segregation, generating four haploid gametes. In the first cell division (meiosis I), homologous chromosomes segregate, whereas, in the second cell division, sister chromatids segregate (meiosis II). For this specialized segregation to occur accurately, several key events including physical connections between homologous chromosomes, sister chromatid cohesion, as well as the monopolar attachment of sister kinetochores (co-orientation of sister kinetochores) at metaphase I  are essential in most organisms (Klein et  al., 1999; Hauf and Watanabe, 2004). Any disturbance in these events may lead to non-disjunction and aneuploidy, or even to nuclear non-reduction in some extreme cases (Watanabe and Nurse, 1999). Homolog pairs must be held together, usually by crossovers (COs), to ensure their alignment on the metaphase I  plate. Crossovers correspond to homologous recombination sites between homologous chromosomes, which can be directly visualized as chiasmata under microscopy. Homologous recombination is initiated by formation of DNA double-strand breaks (DSBs), which are created by the conserved endonuclease

SPO11 (Keeney et al., 1997), and further processed by a number of additional proteins known as the Mre11–Rad50–Xrs2 (MRX) complex (Puizina et al., 2004; Osman et al., 2011). Through two distinct repair pathways, DSBs can give rise either to COs, in which reciprocal exchanges between homolog pairs have occurred, or non-crossovers (NCO), in which reciprocal exchanges have not occurred (Allers and Lichten, 2001; Borner et al., 2004). The physical connection between homologs counteracts the pulling force from the spindle microtubules (MTs), generating the tension required for stabilization of the kinetochore-microtubule attachment (Nicklas, 1997). In the absence of crossovers/chiasmata, homologs segregate randomly at anaphase I, which results in the formation of aneuploid dyads followed by the separation of sister chromatids at anaphase II to generate aneuploid tetrads (de Vries et al., 1999; Chen et al., 2005; Shen et al., 2012). 1 To whom correspondence should be addressed. E-mail zkcheng@genetics. ac.cn, tel. +86-10–6480, 6551, fax +86-10–6480, 6595. 2

These authors contributed equally to this work.

© The Author 2012. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sss145, Advance Access publication 8 December 2012 Received 5 October 2012; accepted 23 November 2012

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Two different classes of COs form through alternative CO formation pathways, including class  I  COs (exhibiting crossover interference, whereby the occurrence of one CO interferes with the occurrence of a proximal CO) and class II COs (interference-independent crossovers, which are randomly distributed). Both classes co-exist in most eukaryotes (Macaisne et  al., 2011; Osman et  al., 2011). The ZMM proteins (Zip1, Zip2, Zip3, Zip4, Msh4, Msh5, Mer3, and Spo16), first identified in Saccharomyces cerevisiae and conserved in most eukaryotes, are essential for the formation of class I COs (Lynn et al., 2007; Youds and Boulton, 2011). Analysis of ZMM mutants showed dramatically reduced CO frequency and loss of interference in budding yeast (Ross-Macdonald and Roeder, 1994; Tsubouchi et  al., 2006), C.  elegans (Kelly et  al., 2000), mammals (Neyton et  al., 2004), and plants (Higgins et  al., 2008; Wang et al., 2009). However, not all organisms utilize both types of COs. The two extreme examples are C. elegans (Zalevsky et  al., 1999), which has only interfering COs, and Schizosaccharomyces pombe with only non-interfering COs (Osman et al., 2003). Neither the mechanism controlling the two classes of CO formation nor the functional relationship between them has been fully characterized. Msh4 and Msh5 (two ZMM proteins) play important roles in promoting crossover formation in eukaryotes. In S. cerevisiae, diploids lacking Msh4 and Msh5 display a reduction in crossover formation and a resultant increase in non-disjunction between homologous chromosomes (Ross-Macdonald and Roeder, 1994; Hollingsworth et al., 1995). In C. elegans, the msh5 mutant shows a dramatic reduction in crossover number compared with the wild-type (Kelly et al., 2000). In Msh4 and Msh5 knockout mice, homologous chromosomes fail to pair correctly and crossovers are completely absent, leading to both male and female sterility (de Vries et  al., 1999; Edelmann et al., 1999). In Arabidopsis, the chiasma frequencies in Atmsh4 and Atmsh5 (T-DNA insertional mutant) are greatly reduced to approximately 15% and 13% of the wild-type level, respectively (Higgins et al., 2004, 2008). Msh4 and Msh5 are known to form a heterodimer stabilizing sdHjs, facilitating resolution as COs and simultaneously imposing crossover interference, and are proposed to act as a sliding clamp embracing the homologous chromosomes (Snowden et al., 2004; Higgins et al., 2008). Interestingly, Msh4 and Msh5 are not found in S. pombe or in Drosophila melanogaster. In S. pombe, crossover formation relies solely on the structurespecific endonuclease complex of Mus81 and Eme1 (Osman et  al., 2003). However, in D.  melanogaster, Msh4 and Msh5 are potentially replaced by other proteins, or an alternative mechanism exists to perform a similar function. Therefore, the roles of Msh4 and Msh5 in meiosis have diverged among different organisms. As one of the most important food crops in the world, rice (Oryza sativa) is also becoming a model monocot for molecular biological studies. Since the completion of the rice genome sequence, functional genomic studies have also become very popular. Several advantages to the use of rice

for investigation of meiosis, including the ease of collecting meiocyte samples, generating meiotic mutants and conducting genetic transformation, make rice the third most-used model plant species for study of the molecular mechanisms of meiosis, following maize and Arabidopsis. In the present study, we cloned OsMSH5 in rice (O. sativa) and investigated its roles during CO formation and synapsis. It was observed that loss of OsMSH5 function resulted in a severe reduction in CO formation.

RESULTS Characterization of a Sterile Mutant A completely sterile mutant was identified in an indica rice variety, Zhongxian 3037, induced by 60Co-γ ray radiation. The mutant exhibited normal plant architecture and could not be distinguished from the wild-type based on the plant morphology during the vegetative stage (Figure  1A and B). However, the pollen grains of the mutant were found to be empty and shrunken at the flowering stage (Figure 1C and D). Moreover, when flowers of the mutant plant were pollinated with wild-type pollens, no seeds were set, suggesting that the megagametogenesis was also affected by this mutation.

Map-Based Cloning of the Gene Related to the Mutation A map-based cloning strategy was used to isolate the mutated gene. As the mutant plant exhibited both male and female sterility, heterozygous plants related to this mutation were crossed with Zhonghua 11, a japonica rice variety. A total of 1485 F2 segregates exhibiting the complete sterile phenotype was used for map-based cloning. Linkage analysis mapped the mutated gene between two STS markers, P1 and P2, on the long arm of chromosome 5, which was further limited to a 55-kb region between other two markers, P3 and P4. Within this 55-kb region, one candidate gene (05g41880) showed high similarity with MSH5 from mice, budding yeast, C.  elegans, and Arabidopsis. The mutants related to MSH5 displayed a reduction in fertility in these organisms. Thus, this candidate gene in the mutant was selected for amplification and sequencing. A single nucleotide T deletion was identified at position 9909, creating a new stop codon in exon 29. Therefore, this sterile mutant was designated Osmsh5-1 (Figure 2). Four additional mutants exhibiting the same defects as Osmsh5-1 treated with 60Co-γ ray radiation were obtained: two from Zhongxian 3037 and one each from Wuxiangjing 9 and Nipponbare. The mutated genes in these four mutants were independently mapped to a similar chromosome region containing OsMSH5 by the same mapping strategy. Further sequencing analysis revealed that OsMSH5 genes in these four mutants had different mutations, one with nucleotides ATT (8893–8895) deleted in exon 27 resulting in a downstream frameshift, one with an A to G (nucleotide 9897) point

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Figure 1.  The Rice Osmsh5-1 Mutant Phenotype. (A) Comparison of a wild-type (WT) plant (left) and an Osmsh5-1 mutant plant (right). (B) Comparison of a WT panicle (left) and an Osmsh5-1 mutant panicle (right). (C) Viable pollen grains in a WT plant. (D) Inviable pollen grains in an Osmsh5-1 mutant plant.Scale bars = 50 μm.

mutation within exon 29 causing a histidine to arginine substitution at amino acid 686, one with a G to A  (nucleotide 7884, the splicing site) mutation in intron 21, and one with a G to A  (nucleotide 3175)  point mutation within exon 10 leading to an arginine to lysine substitution at amino acid 237. Thus, these alleles were designated Osmsh5-2, Osmsh5-3, Osmsh5-4, and Osmsh5-5, respectively (Figure 2). As Osmsh5-1 is a deletion line creating a new stop codon, we selected it for further cytological investigation.

The Structure of OsMSH5 and Its Protein Sequence The full-length cDNA sequence of OsMSH5, identified by RT–PCR and rapid amplification of cDNA ends (RACE) PCR, is

consistent with AK101127 from the NCBI website. The gene comprises 34 exons and 33 introns and its open reading frame encodes a predicted protein consisting of 809 amino acids. OsMSH5 shows significant homology with MSH5 in Arabidopsis (66% identity and 82% similarity) and is also highly related to the MSH5 in mammals and budding yeast (Supplemental Figure  1). OsMSH5 contains a conserved ATP-binding motif at its C-terminus (amino acids: 538–746, conserved domains search in NCBI; www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi), and is similar to AtMSH5 lacking the MutS domain I, which is essential for the activity of DNA mismatch repairing. By means of RT–PCR, we found that OsMSH5 is expressed mainly in young panicles and to lesser extent in internodes,

Figure 2.  Schematic Representation of the OsMSH5 Gene and Mutation Sites in Osmsh5 Alleles. Coding regions are shown as black boxes; untranslated regions are shown as lines. The triangles indicate the mutation sites in different Osmsh5 mutants.

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roots, and leaves (Supplemental Figure  2). In addition, it was detected that the expression of OsMSH5 is dramatically reduced in Osmsh5-1 (Supplemental Figure 2).

Meiosis Is Disrupted in Osmsh5 To analyze the cause of sterility in Osmsh5 mutants, chromosome behavior in pollen mother cells (PMCs) at different stages of meiosis were investigated in both wild-type and Osmsh51 meiocytes. In the wild-type, generation of single-strand threads from meiotic chromosomes was first observed at leptotene (Figure  3A). At zygotene, chromosomes congregated into a small volume and wrapped around the nucleolus on one side. Homologous chromosome pairing and synapsis were first observed during this stage (Figure 3B). Full chromosome synapsis was reached at pachytene (Figure 3C). Paired chromosomes condensed into 12 rod-like or ring-like bivalents at diakinesis (Figure  3D). On entry into metaphase I, 12 extremely condensed bivalents aligned on the equatorial plate (Figure 3E). Subsequently, homologous chromosomes separated equally and migrated to opposite poles of the cell (Figure 3F), leading to dyad formation (Figure 3G). During the second meiotic division, sister chromatids of each chromosome segregated, with tetrad formation at the end of meiosis (Figure 3H and I). In the Osmsh5-1 mutant, meiotic chromosome behavior was almost identical to that of the wild-type from leptotene to pachytene (Figure 3J and L). However, several chromosomal defects were apparent from diakinesis through to the following meiotic stages. Different numbers of univalents were observed at diakinesis in Osmsh5-1 (Figure 3M). At metaphase I, all bivalents aligned on the equatorial plate, while univalents were dispersed within the nucleus (Figure  3N). During anaphase I, unequal chromosome segregation was detected (Figure 3O). The two homologous chromosomes of the bivalents separated and moved to the opposite poles of the cell, as did those of the wild-type. However, two different segregation modes of the univalents were observed in the same cell. One mode involved monopolar centromere attachment, resulting in movement of the univalent to one pole of the cell, while the second mode involved bipolar centromere attachment, leading to early separation of the two chromatids and movement to the opposite poles (Figure 3P). The second meiotic division occurred subsequently and resulted in the formation of tetrads with aneuploid numbers of chromosomes accompanied by several micronuclei (Figure 3Q and R). We also investigated chromosome behaviors in Osmsh5-2, Osmsh5-3, Osmsh5-4, and Osmsh5-5 meiocytes; they were pretty similar to those in Osmsh5-1 (Supplemental Figure 3A–3D).

OsMSH5 Is a Meiosis-Specific Protein To define the spatial and temporal distribution of OsMSH5 during meiosis, dual immunolocalization was performed on wild-type microsporocytes using polyclonal antibodies specific for OsREC8 and OsMSH5, raised in rabbit and mouse, respectively. In wild-type, OsMSH5 foci appeared almost at the same time as OsREC8 signals at leptotene (Figure 4A). The

number of OsMSH5 foci accumulated rapidly and reached a peak at early zygotene (Figure 4B). At early pachytene, most OsMSH5 foci started to leave from chromosomes (Figure 4C). With the progression of meiosis, however, the number of OsMSH5 foci decreased rapidly and only a few residual foci were observed at late pachytene (Figure 4D). OsMSH5 signals were absent at diplotene and could not be detected thereafter. Immunolocalization studies were also performed on Osmsh5-1 PMCs. No OsMSH5 signals were detected in its meiocytes, even in late leptotene/early zygotene during which OsMSH5 foci were normally the most prominent, showing Osmsh5-1 to be a null mutant (Supplemental Figure 4).

OsMSH5 Is Required for the Normal Crossover Formation From diakinesis to metaphase I, the obvious cytological defect in Osmsh5-1 PMCs was the presence of univalents; and the number of bivalents was greatly reduced (Figure 3M and N). The mean bivalent frequency in Osmsh5-1 was reduced to 2.03 per cell, in contrast to 12 per cell in the wild-type. In fact, no more than four bivalents were detected in all the examined mutant metaphase I PMCs (n = 97). Chiasmata play an important role in the formation of stable bivalents and, therefore, the chiasma frequency at diakinesis was quantified in both Zhongxian 3037 and Osmsh5-1 using previously described criteria (Sanchez-Moran et al., 2001). The rod-shaped bivalent was scored as having one chiasma, while the ring-shaped bivalent was scored as having two chiasmata. In the wild-type, the chiasma frequency was 19.38 ± 1.66 per cell (n  =  77) compared with 2.10 ± 0.91 per cell in Osmsh51 (n  =  97). Thus, the number of chiasmata was reduced dramatically in Osmsh5-1 and further affected the subsequent formation of bivalents at both diakinesis and metaphase I.  The distribution of the remaining chiasmata was analyzed in Osmsh5-1 and compared with the corresponding data in the wild-type. Statistical analysis showed that the chiasma distribution per chromosome in the wild-type deviated significantly from a Poisson distribution (Supplemental Figure 5A; χ[3]2 = 526.5, P < 0.001), while the distribution of residual chiasmata on chromosomes in Osmsh5-1 was consistent with a predicted Poisson distribution (Supplemental Figure 5B; χ[3]2 = 5.2, P > 0.05). These results suggested that the majority of the residual chiasmata were randomly distributed on chromosomes in Osmsh5-1 and that the mutation of OsMSH5 disrupted the formation of interference-sensitive COs dramatically.

CO Frequencies in Double Mutants of Osmsh5-1 with Other zmm Mutants Are Similar to that in Osmsh5-1 We generated three double mutants, including Osmsh5 Oszip4, Osmsh5 Osmer3, and Osmsh5 hei10, by crossing the heterozygous plant Osmsh5-1+/– with three other heterozygous plants, Oszip4+/–, Osmer3+/–, and hei10+/–, respectively, and identified them by individual genotyping analysis of the F2 populations. The chiasma frequencies at metaphase I of those double mutants as well as their corresponding single mutants

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Figure 3.  Meiotic Chromosome Behaviors of PMCs in Both Wild-Type and the Osmsh5-1 Mutant. (A–I) The wild-type. (J–R) The Osmsh5-1 mutant. (A, J) Leptotene. (B, K) Zygotene. (C, L) Pachytene. (D, M) Diakinesis. (E, N) Metaphase I. (F, O) Anaphase I. (G, P) Telophase I. (H, Q) Metaphase II. (I, R) Tetrads. Chromosomes are stained with DAPI. Scale bars = 5 μm.

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Figure 4.  Dual Immunostaining of OsREC8 and OsMSH5 in Wild-Type Meiocytes. (A) Leptotene. (B) Zygotene. (C) Early pachytene. (D) Pachytene. An anti-OsREC8 antibody is used to indicate the meiotic chromosomes (red). Scale bars = 5 μm.

were investigated using the same described criteria (Table 1). We found the chiasma number in the three double mutants all ranged from 0 to 4 (Figure 5B, D, and 5F). The mean chiasma frequency of each double mutant was very close to that in Osmsh5-1, but much less than that in the other relative single mutants like Oszip4, Osmer3, and hei10 (Figure  5A, C, and 5E). Therefore, we propose that OsMSH5 functions upstream of OsZIP4, OsMER3, and HEI10 in regulating CO formation.

Meiosis Proceeds Normally and the Synaptonemal Complex (SC) Is Well Installed in Osmsh5 Several early meiotic elements in Osmsh5-1 meiocytes were examined by immunostaining. Meiotic recombination is

initiated by DSBs and completed through their programmed repair from early leptotene to zygotene. To investigate the occurrence of recombination in Osmsh5-1, antibodies specific for the phosphorylated form of the histone variant H2AX (γH2AX) were used to monitor DNA DSBs formation. In Osmsh5-1 PMCs, strong γH2AX immunostaining was visible (Figure  6A), implying that DSB formation was not disturbed and that meiotic recombination was initiated normally. OsAM1 is required for the leptotene–zygotene transition and its deficiency leads to meiosis arrest at leptotene (Che et al., 2011). OsAM1 was found to be normally loaded onto meiotic chromosomes in the Osmsh5-1 mutant (Figure 6B), indicating that OsAM1 acts at an earlier stage in meiosis than OsMSH5.

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Table 1.  Chiasma Frequency in Different Genotypes.  Genotype

Chiasma/cell

Reference

Wild-type

19.38 ± 1.66* (n = 77)

This study

Osmsh5-1

2.10 ± 0.91 (n = 97)

This study

Oszip4

6.05 ± 1.97 (n =164)

Shen et al., 2012

Osmer3

5.59 ± 2.07 (n = 64)

Shen et al., 2012

hei10

6.50 ± 2.10 (n =130)

Wang et al., 2012

Osmsh5 Oszip4

2.07 ± 0.83 (n = 82)

This study

Osmsh5 Osmer3

2.13 ± 0.93 (n = 76)

This study

Osmsh5 hei10

2.06 ± 0.87 (n = 81)

This study

* Mean ± SD. n, number of pollen mother cells observed.

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The pachytene chromosome morphology was also found to be relatively normal in Osmsh5-1 meiocytes (Figure 3L), indicating that synapsis occurred in the mutant. Therefore, the loading of several proteins related to both the lateral and central elements of SCs in Osmsh5-1 was analyzed by dual immunodetection. OsREC8 is a component of the meiotic cohesion complex, which is required for axial element formation and homologous chromosome pairing. OsREC8 loaded normally onto the meiotic chromosomes during prophase I in Osmsh5-1 (Figure  6C), implying the meiotic chromosome axis was well formed. PAIR2 is associated with unpaired axial elements in rice (Nonomura et al., 2006). PAIR2 foci are detected on early zygotene chromosomes and dissociate from chromosomes

Figure 5.  Metaphase I Chromfosomes in Different Single or Double Mutants. (A) Oszip4. (B) Osmsh5 Oszip4. (C) Osmer3. (D) Osmsh5 Osmer3. (E) hei10. (F) Osmsh5 hei10. Chromosomes are stained with DAPI. Scale bars = 5 μm.

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Figure  6.  Immunodetection of γH2AX, OsAM1, PAIR2, PAIR3, ZEP1, OsMER3, and OsZIP4 Antibodies on Meiotic Spreads of Osmsh5-1 Chromosomes.Anti-CENH3 and anti-OsREC8 antibodies are used to indicate PMCs. (A)γH2AX in a leptotene meiocyte. (B) OsAM1 in a zygotene meiocyte. (C) PAIR2 in a zygotene meiocyte. (D) PAIR2 in a pachytene meiocyte. (E) PAIR3 in a pachytene meiocyte. (F) ZEP1 in a pachytene meiocyte. (G) OsMER3 in a zygotene meiocyte. (H) OsZIP4 in a zygotene meiocyte. Scale bars = 5 μm.

as SC assembly is completed during early pachytene. In the Osmsh5-1 mutant, PAIR2 loaded normally onto chromosomes during early zygotene (Figure 6C) and depleted at pachytene, similar to that in the wild-type (Figure 6D). PAIR3 is a meiotic axial element in rice (Wang et  al., 2011), with a localization pattern that is very similar to OsREC8. In Osmsh5-1 meiocytes, PAIR3 localization onto meiotic chromosomes was similar to that of wild-type meiocytes (Figure 6E). ZEP1, which is a transverse filament protein that constitutes the central element of the SC in rice (Wang et  al., 2010), localizes on leptotene chromosomes as punctate foci and forms continuous linear signals along the whole length of chromosomes at pachytene. In Osmsh5-1, punctate ZEP1 immunostaining signals were detected from leptotene through zygotene, and continuous linear signals were observed along pachytene chromosomes, also similar to that in the wild-type (Figure 6F). Taken together, we conclude that the SC is well formed in Osmsh5-1.

The Loading of OsZIP4, OsMER3, and HEI10 Depends on OsMSH5, but Not Vice Versa In addition to ZEP1, three other ZMM proteins including OsZIP4, OsMER3, and HEI10 have been characterized in rice so far (Wang et al., 2009; Shen et al., 2012; Wang et al., 2012). Loss of function of all these proteins leads to similar meiotic defects with dramatically decreased numbers of COs, which is very similar to the defects observed in Osmsh5. Therefore,

expression of the three proteins in Osmsh5-1 was analyzed by immunodetection. OsZIP4 and OsMER3 were not loaded onto meiotic chromosomes in the absence of OsMSH5. Almost no immunostaining of OsZIP4 or OsMER3 was detected in Osmsh5-1 (Figure 6G and H). However, the performance of HEI10 differed from that of OsZIP4 and OsMER3 in Osmsh5-1. In Osmsh5-1, HEI10 foci appeared normally at early leptotene (Figure 7A). At late leptotene, a mean of 287.5 HEI10 foci were observed (n  =  10; range, 210–335). The localization of HEI10 from leptotene to early pachytene was indistinguishable from that of the wildtype (Figure 7B and C). However, prominent HEI10 foci were significantly reduced in Osmsh5-1 (Figure 7D), with an average of 12.2 (n = 22; range, 10–15) in Osmsh5-1 compared with 24.5 in the wild-type. During diakinesis, a few HEI10 foci were randomly distributed in the nucleoplasm, while none was located on the remaining bivalents (n = 22) (Figure 7E). No HEI10 foci were detected after metaphase I  (Figure  7F). Based on our observations, it can be speculated that HEI10 is normally loaded onto meiotic chromosomes before early pachytene. However, the formation of prominent foci at late pachytene and their stable maintenance onto chromosomes after diplotene were significantly disrupted in the absence of OsMSH5. Immunodetection of OsMSH5 was also performed in hei10, Oszip4, and Osmer3 meiocytes to further characterize the relationship among these proteins. It was observed that OsMSH5

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Figure 7.  The Localization of HEI10 in the Osmsh5-1 Mutant. (A) Leptotene. (B) Zygotene. (C) Early pachytene. (D) Late pachytene. (E) Diakinesis, the prominent HEI10 foci scatter in the cytoplasm. (F) Metaphase I. Chromosomes are stained with DAPI (blue). Scale bars = 5 μm.

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Figure 8.  Immunofluorescent Localization of OsMSH5 in Prophase I Meiocytes in Different zmm Mutants. (A) hei10. (B) Oszip4. (C) Osmer3. The anti-OsREC8 antibody (red) is used to indicate the meiotic chromosomes. Scale bars = 5 μm.

proteins were loaded normally onto the chromosomes and behaved in a similar manner to that of wild-type meiocytes at meiotic prophase I in hei10, Oszip4, and Osmer3 mutants (Figure 8), implying that the normal loading of OsMSH5 is independent of HEI10, OsZIP4, and OsMER3, which is consistent with the above hypothesis drawn by counting the residual bivalents among different zmm mutants that OsMSH5 may function upstream of HEI10, OsZIP4, and OsMER3 in regulating CO formation.

Discussion OsMSH5 May Function as an Early Element among the ZMM Complex In budding yeast, a group of proteins called ZMM proteins (Zip1, Zip2, Zip3, Zip4, Msh4, Msh5, Mer3, and Spo16) are implicated in CO formation (Lynn et al., 2007; Youds and Boulton, 2011). Mutants of all these genes and their double mutants exhibit a similar reduction in CO formation to approximately 15% of the wild-type level, suggesting that these proteins act in the same pathway, presumably as components of a protein complex. This residual CO formation is consistent with studies suggesting that there are two classes of COs in budding yeast (de los Santos et al., 2003; Hollingsworth and Brill, 2004). Most COs

arise from the ZMM-dependent class I pathway and exhibit CO interference, while the remaining of COs (10–20%), referred to as class II COs, depend on the activity of the MUS81/MMS4 heterodimer and do not exhibit interference (Hollingsworth and Brill, 2004). Among the ZMM complex proteins, Zip1 is a transverse filament protein of SCs (Sym et al., 1993). Zip2 is related to the WD40-like repeat protein (Chua and Roeder, 1998). Zip3 is a RING domain-containing protein that may have ubiquitin or ubiquitin-like modifier (SUMO) ligase activity (Agarwal and Roeder, 2000). Zip4 is a tetra-tricopeptide repeat (TPR) protein (Tsubouchi et al., 2006). Furthermore, Zip2, Zip3, and Zip4 proteins are involved in ubiquitinylation and/or SUMOylation and are expected to function cooperatively to modify protein interactions. Mer3 is a DNA helicase that unwinds various duplex DNA in an ATP-dependent manner (Nakagawa et  al., 2001). MSH4 and MSH5 appear to function as a heterodimer to stabilize strand invasion (Snowden et al., 2004). Several presumed ZMM homologs have been identified in plants, indicating conservation of the ZMM group polypeptides among different species. MER3, MSH4, and MSH5 are highly conserved proteins and their orthologs have been identified and functionally investigated in plants. Despite poor sequence conservation, the Zip1 homologs, ZYP1 and

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ZEP1, have been isolated in Arabidopsis and rice, respectively (Higgins et  al., 2005; Wang et  al., 2010). However, mutation of either ZYP1 or ZEP1 does not lead to obvious reduction in COs, indicating different roles during CO formation. Presumed Zip4 orthologs have been identified in Arabidopsis and rice, independently (Chelysheva et al., 2007; Shen et al., 2012). SHOC1 has been identified as the ortholog of Zip2 (Macaisne et  al., 2008). In addition, a novel gene, PARTING DANCERS (PTD), is found to be epistatic to ZMMs in Arabidopsis (Wijeratne et  al., 2006). More recently, Zip3 orthologs have been identified in both Arabidopsis and rice (Chelysheva et al., 2012; Wang et al., 2012). However, the relationship among different components of the ZMM complex has not been thoroughly investigated, especially their biological roles in class  I  crossover formation. In the present study, we have carefully investigated those aspects by dual immunofluorescence staining using ZMM protein-specific antibodies. OsMSH5 was shown to be loaded normally onto meiotic chromosomes in Oszip4 and Osmer3, while localizations of OsZIP4, OsMER3, and HEI10 all were found to be severely affected in Osmsh51, suggesting that OsMSH5 functions upstream of OsZIP4, OsMER3, and HEI10. This hypothesis is also supported by quantification of the remaining COs in mutants, such as Osmsh5, zip4, mer3, and hei10. COs in Osmsh5 were the least frequent among the zmm mutants, with only 2.03 in Osmsh5-1. However, COs were detected at frequencies of 6.05, 5.59, and 6.50 in Oszip4, Osmer3, and hei10, respectively. Nonetheless, COs in the double mutant Oszip4 Osmer3 (3.13) were also more frequent than that in Osmsh51 (Shen et  al., 2012). It can be speculated that blockade of the pathway of class I COs results from loss function of OsMSH5, while depletion of other ZMM components such as OsZIP4, OsMER3, and HEI10 leads only to partial disruption of this pathway in rice. It was also observed that the SC is well installed in Osmsh5 and that OsMSH5 localizes normally on meiotic chromosomes of zep1, suggesting that OsMSH5 and ZEP1 play independent roles in both CO formation and SC installation, which is also conserved in Arabidopsis (Higgins et al., 2008).

The Late Recombination Nodules Are Affected in the Absence of OsMSH5 The specific mechanism governing CO distribution is still primordial. Cytologically, CO distribution can be directly shown by recombination nodules. There are two kinds of recombination nodules formed in the course of CO formation, including early nodule (EN) and late nodule (LN) (Dawe, 1998; Page and Hawley, 2004). ENs represent the initial recombination sites, while LNs indicate COs that are assumed to mature into chiasmata. Only a small number of ENs are likely to be converted into final LNs. A number of proteins have been identified to be related to LNs in different organisms. In budding yeast, ZMM foci correspond to LNs and are considered to mark the

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final class I COs sites (Fung et al., 2004; Tsubouchi et al., 2008). In C.  elegans, the Zip3 homolog ZHP-3 appeared in prominent foci and localized to CO sites from late pachytene into diplotene stages (Bhalla et al., 2008). In mice, both MLH1 and MLH3 foci at the pachytene stage were used to mark the CO positions (Lipkin et  al., 2002; Marcon and Moens, 2003). In Arabidopsis, foci of MLH1, MLH3, and HEI10 all correspond to CO sites (Jackson et  al., 2006; Chelysheva et  al., 2012). However, in rice, HEI10 is the only protein that has been identified to be able to show CO sites. In this study, we found HEI10 foci appeared normally at early leptotene in Osmsh5-1. And the localization of HEI10 from leptotene to early pachytene was also indistinguishable from that of the wild-type. However, the prominent HEI10 foci were significantly reduced in Osmsh5-1(about half of the wild-type), with an average of 12.2 in Osmsh5-1 compared with 24.5 in the wild-type (Wang et  al., 2012). We suspect that the late recombination nodule determination at late pachytene and their stable maintenance onto chromosomes channeling them irreversibly towards CO maturation after diplotene both rely on the function of OsMSH5.

METHODS Plant Materials Osmsh5-1, Osmsh5-2, and Osmsh5-4 were identified from an indica rice variety, Zhongxian 3037, induced by 60Co~γ ray radiation. Osmsh5-3 and Osmsh5-4 were identified from two japonica rice varieties, Wuxiangjing 9 and Nipponbare, respectively, also induced by 60Co~γ ray radiation. Both Zhongxian 3037 and another japonica rice variety, Zhonghua 11, were used as polymorphic varieties to make mapping populations by crossing with the heterozygous plants of those Osmsh5 mutants. Four other meiotic mutants, including Oszip4, Osmer3, hei10, and zep1, were described previously (Wang et al., 2009, 2010; Shen et al., 2012; Wang et al., 2012). All plants were grown in the paddy fields under normal growth conditions.

Molecular Cloning of OsMSH5 STS markers were developed for mapping of OsMSH5 based on sequence differences between japonica variety Nipponbare and indica variety 93–11 from the data published on the NCBI website. Primers were designed using the PrimerSelect program of Lasergene (DNASTAR, Madison, WI). All primers used in this study are listed in Supplemental Table 1.

RT–PCR Analysis Total RNA was isolated from roots, stem, leaves, and young panicles using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. 4 μg RNA was reverse-transcribed with Oligo-dT(18) primer using M-MLV reverse transcriptase (Promega). The primer pairs P5 were designed to examine the expression of OsMSH5 in the wild-type. The standard control Ubiquitin gene was examined with the primer pairs P6.

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RT–PCR analysis was performed with the following profile: 94°C for 3 min; 25–30 cycles at 94°C for 30 s, 57°C for 30 s and 72°C for 30 s; and 72°C for 10 min.

Computational and Database Analysis The gene structure schematic diagram was drawn using GSDS (http://gsds.cbi.pku.edu.cn/index.php) and further edited with Adobe Illustrator CS5. Protein sequence similarity searches were performed at the NCBI (www.ncbi.nlm. nih.gov/BLAST). The alignment of amino acid sequences was performed with the MEGA3.1 software (www.megasoftware.net/), and further modified using the Alignx module of Vector NTI Advance 9.0 (Invitrogen). Domain searches were performed using the HMMER-based SMART website (http:// smart.embl-heidelberg.de/).

Antibodies To generate the antibody against OsMSH5, a 300-bp fragment of OsMSH5 cDNA was amplified using primer pairs P7. The fragment was ligated into the expression vector pGEX4T-2 (Amersham) digested with BamHI–EcoRI. The expression vectors were transformed into BL21 (DE3) and induced by addition of 0.2  mM IPTG to the culture medium. The fusion peptides were expressed in a soluble fraction and purified using the glutathione Sepharose 4B (GE). Polyclonal antibodies of OsMSH5 were raised against in mouse. The OsAM1, OsREC8, PAIR2, PAIR3, OsMER3, OsZIP4, ZEP1, and HEI10 antibodies used in this study were generated previously (Wang et  al., 2009, 2010; Che et  al., 2011; Shao et  al., 2011; Wang et  al., 2011; Shen et al., 2012).

Meiotic Chromosome Preparation and Immunofluorescence Young panicles of both wild-type and mutants were harvested and fixed in Carnoy’s solution (ethanol:glacial acetic acid, 3:1) and stored at –20°C. Microsporocytes at the meiotic stage were squashed and stained with acetocarmine. Slides with chromosome preparations were frozen in liquid nitrogen. After removing the coverslips, the slides were dehydrated through an ethanol series (70%, 90%, and 100%). Chromosomes were counterstained with 4,6-diamidinophenylindole (DAPI) in an antifade solution (Vector Laboratories, Burlingame, CA). Chromosome images were captured under the Olympus BX61 fluorescence microscope with a microCCD camera. For immunodetection, fresh young panicles were fixed in 4% (w/v) paraformaldehyde for 30 min at room temperature. Chromosome preparation and immunofluorescence were carried out as previously described (Wang et al., 2009).

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This work was supported by grants from the Ministry of Sciences and Technology of China (2011CB944602 and 2012AA10A301), the State Key Laboratory of Plant Genomics of China (2012A0527), and the National Natural Science Foundation of China (31160223 and 31230038). No conflict of interest declared.

References Agarwal, S., and Roeder, G.S. (2000). Zip3 provides a link between recombination enzymes and synaptonemal complex proteins. Cell. 102, 245–255. Allers, T., and Lichten, M. (2001). Differential timing and control of noncrossover and crossover recombination during meiosis. Cell. 106, 47–57. Bhalla, N., Wynne, D.J., Jantsch, V., and Dernburg, A.F. (2008). ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans. PLoS Genet. 4, e1000235. Borner, G.V., Kleckner, N., and Hunter, N. (2004). Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis. Cell. 117, 29–45. Che, L., Tang, D., Wang, K., Wang, M., Zhu, K., Yu, H., Gu, M., and Cheng, Z. (2011). OsAM1 is required for leptotene–zygotene transition in rice. Cell Res. 21, 654–665. Chelysheva, L., Gendrot, G., Vezon, D., Doutriaux, M.P., Mercier, R., and Grelon, M. (2007). Zip4/Spo22 is required for class I CO formation but not for synapsis completion in Arabidopsis thaliana. PLoS Genet. 3, e83. Chelysheva, L., Vezon, D., Chambon, A., Gendrot, G., Pereira, L., Lemhemdi, A., Vrielynck, N., Le Guin, S., Novatchkova, M., and Grelon, M. (2012). The Arabidopsis HEI10 is a new ZMM protein related to Zip3. PLoS Genet. 8, e1002799. Chen, C., Zhang, W., Timofejeva, L., Gerardin, Y., and Ma, H. (2005). The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation. Plant J. 43, 321–334. Chua, P.R., and Roeder, G.S. (1998). Zip2, a meiosis-specific protein required for the initiation of chromosome synapsis. Cell. 93, 349–359. Dawe, R.K. (1998). Meiotic chromosome organization and segregation in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 371–395. de los Santos, T., Hunter, N., Lee, C., Larkin, B., Loidl, J., and Hollingsworth, N.M. (2003). The Mus81/Mms4 endonuclease acts independently of double-Holliday junction resolution to promote a distinct subset of crossovers during meiosis in budding yeast. Genetics. 164, 81–94. de Vries, S.S., Baart, E.B., Dekker, M., Siezen, A., de Rooij, D.G., de Boer, P., and te Riele, H. (1999). Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male and female meiosis. Genes Dev. 13, 523–531.

Luo et al.  •  OsMSH5 Cloning   

Edelmann, W., Cohen, P.E., Kneitz, B., Winand, N., Lia, M., Heyer, J., Kolodner, R., Pollard, J.W., and Kucherlapati, R. (1999). Mammalian MutS homologue 5 is required for chromosome pairing in meiosis. Nat. Genet. 21, 123–127. Fung, J.C., Rockmill, B., Odell, M., and Roeder, G.S. (2004). Imposition of crossover interference through the nonrandom distribution of synapsis initiation complexes. Cell. 116, 795–802. Hauf, S., and Watanabe, Y. (2004). Kinetochore orientation in mitosis and meiosis. Cell. 119, 317–327. Higgins, J.D., Armstrong, S.J., Franklin, F.C., and Jones, G.H. (2004). The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis. Genes Dev. 18, 2557–2570. Higgins, J.D., Sanchez-Moran, E., Armstrong, S.J., Jones, G.H., and Franklin, F.C. (2005). The Arabidopsis synaptonemal complex protein ZYP1 is required for chromosome synapsis and normal fidelity of crossing over. Genes Dev. 19, 2488–2500. Higgins, J.D., Vignard, J., Mercier, R., Pugh, A.G., Franklin, F.C., and Jones, G.H. (2008). AtMSH5 partners AtMSH4 in the class I meiotic crossover pathway in Arabidopsis thaliana, but is not required for synapsis. Plant J. 55, 28–39. Hollingsworth, N.M., and Brill, S.J. (2004). The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions. Genes Dev. 18, 117–125. Hollingsworth, N.M., Ponte, L., and Halsey, C. (1995). MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 9, 1728–1739. Jackson, N., Sanchez-Moran, E., Buckling, E., Armstrong, S.J., Jones, G.H., and Franklin, F.C. (2006). Reduced meiotic crossovers and delayed prophase I  progression in AtMLH3-deficient Arabidopsis. EMBO J. 25, 1315–1323. Keeney, S., Giroux, C.N., and Kleckner, N. (1997). Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell. 88, 375–384. Kelly, K.O., Dernburg, A.F., Stanfield, G.M., and Villeneuve, A.M. (2000). Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis. Genetics. 156, 617–630. Klein, F., Mahr, P., Galova, M., Buonomo, S.B., Michaelis, C., Nairz, K., and Nasmyth, K., (1999). A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell. 98, 91–103. Lipkin, S.M., Moens, P.B., Wang, V., Lenzi, M., Shanmugarajah, D., Gilgeous, A., Thomas, J., Cheng, J., Touchman, J.W., Green, E.D., et al. (2002). Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat. Genet. 31, 385–390. Lynn, A., Soucek, R., and Borner, G.V. (2007). ZMM proteins during meiosis: crossover artists at work. Chromosome Res. 15, 591–605. Macaisne, N., Novatchkova, M., Peirera, L., Vezon, D., Jolivet, S., Froger, N., Chelysheva, L., Grelon, M., and Mercier, R. (2008). SHOC1, an XPF endonuclease-related protein, is essential for the formation of class  I  meiotic crossovers. Curr. Biol. 18, 1432–1437.

741

Macaisne, N., Vignard, J., and Mercier, R. (2011). SHOC1 and PTD form an XPF-ERCC1-like complex that is required for formation of class I crossovers. J. Cell Sci. 124, 2687–2691. Marcon, E., and Moens, P. (2003). MLH1p and MLH3p localize to precociously induced chiasmata of okadaic-acid-treated mouse spermatocytes. Genetics. 165, 2283–2287. Nakagawa, T., Flores-Rozas, H., and Kolodner, R.D. (2001). The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3’ to 5’ direction. J. Biol. Chem. 276, 31487–31493. Neyton, S., Lespinasse, F., Moens, P.B., Paul, R., Gaudray, P., PaquisFlucklinger, V., and Santucci-Darmanin, S. (2004). Association between MSH4 (MutS homologue 4)  and the DNA strandexchange RAD51 and DMC1 proteins during mammalian meiosis. Mol. Hum. Reprod. 10, 917–924. Nicklas, R.B. (1997). How cells get the right chromosomes. Science. 275, 632–637. Nonomura, K., Nakano, M., Eiguchi, M., Suzuki, T., and Kurata, N. (2006). PAIR2 is essential for homologous chromosome synapsis in rice meiosis I. J. Cell Sci. 119, 217–225. Osman, F., Dixon, J., Doe, C.L., and Whitby, M.C. (2003). Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81–Eme1 in meiosis. Mol. Cell. 12, 761–774. Osman, K., Higgins, J.D., Sanchez-Moran, E., Armstrong, S.J., and Franklin, F.C. (2011). Pathways to meiotic recombination in Arabidopsis thaliana. New Phytol. 190, 523–544. Page, S.L., and Hawley, R.S. (2004). The genetics and molecular biology of the synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20, 525–558. Puizina, J., Siroky, J., Mokros, P., Schweizer, D., and Riha, K. (2004). Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis. Plant Cell. 16, 1968–1978. Ross-Macdonald, P., and Roeder, G.S. (1994). Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction. Cell. 79, 1069–1080. Sanchez-Moran, E., Armstrong, S.J., Santos, J.L., Franklin, F.C., and Jones, G.H. (2001). Chiasma formation in Arabidopsis thaliana accession Wassileskija and in two meiotic mutants. Chromosome Res. 9, 121–128. Shao, T., Tang, D., Wang, K., Wang, M., Che, L., Qin, B., Yu, H., Li, M., Gu, M., and Cheng, Z. (2011). OsREC8 is essential for chromatid cohesion and metaphase I monopolar orientation in rice meiosis. Plant Physiol. 156, 1386–1396. Shen, Y., Tang, D., Wang, K., Wang, M., Huang, J., Luo, W., Luo, Q., Hong, L., Li, M., and Cheng, Z. (2012). The role of ZIP4 in homologous chromosome synapsis and crossover formation in rice meiosis. J. Cell Science. 125, 2581–2591. Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004). hMSH4-hMSH5 recognizes Holliday Junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes. Mol. Cell. 15, 437–451. Sym, M., Engebrecht, J.A., and Roeder, G.S. (1993). ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis. Cell. 72, 365–378.

742 

  Luo et al.  •  OsMSH5 Cloning

Tsubouchi, T., Macqueen, A.J., and Roeder, G.S. (2008). Initiation of meiotic chromosome synapsis at centromeres in budding yeast. Genes Dev. 22, 3217–3226. Tsubouchi, T., Zhao, H., and Roeder, G.S. (2006). The meiosis-specific zip4 protein regulates crossover distribution by promoting synaptonemal complex formation together with zip2. Dev. Cell. 10, 809–819.

Wang, M., Wang, K., Tang, D., Wei, C., Li, M., Shen, Y., Chi, Z., Gu, M., and Cheng, Z. (2010). The central element protein ZEP1 of synaptonemal complex regulates the number of crossovers during meiosis in rice. Plant Cell. 22, 417–430. Watanabe, Y., and Nurse, P. (1999). Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature. 400, 461–463.

Wang, K., Tang, D., Wang, M., Lu, J., Yu, H., Liu, J., Qian, B., Gong, Z., Wang, X., Chen, J., et al. (2009). MER3 is required for normal meiotic crossover formation, but not for presynaptic alignment in rice. J. Cell Sci. 122, 2055–2063.

Wijeratne, A.J., Chen, C., Zhang, W., Timofejeva, L., and Ma, H. (2006). The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination. Mol. Biol. Cell. 17, 1331–1343.

Wang, K., Wang, M., Tang, D., Shen, Y., Miao, C., Hu, Q., Lu, T., and Cheng, Z. (2012). The role of rice HEI10 in the formation of meiotic crossovers. PLoS Genet. 8, e1002809.

Youds, J.L., and Boulton, S.J. (2011). The choice in meiosis-defining the factors that influence crossover or non-crossover formation. J. Cell Sci. 124, 501–513.

Wang, K., Wang, M., Tang, D., Shen, Y., Qin, B., Li, M., and Cheng, Z. (2011). PAIR3, an axis-associated protein, is essential for the recruitment of recombination elements onto meiotic chromosomes in rice. Mol. Biol. Cell. 22, 12–19.

Zalevsky, J., MacQueen, A.J., Duffy, J.B., Kemphues, K.J., and Villeneuve, A.M. (1999). Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast. Genetics. 153, 1271–1283.