Genetic collection of meiotic mutants of rye Secale cereale L

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F2 generations after self-fertilization of the F1 hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome "bouquet" was impaired, and all chromosomes were univalent in meiotic metaphase I in 96% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase II. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Genetic Collection of Meiotic Mutants of Rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F₂ generations after self-fertilization of the F₁ hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome “bouquet” was impaired, and all chromosomes were univalent in meiotic metaphase I in 96.8% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase I. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with 14 univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei8-10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Genetic analysis of inheritance of mei8, sy1 and sy10 mutations,disrupting the correct meiosis in the rye Secale cereale L

It is shown that mutations mei8 (irregular condensation and fragmentation of meiotic chromosomes), sy1 (asynapsis), and sy10 (heterologous synapsis) of rye Secale cereal are nonallelic. In double mutants mei8 sy1 and mei8 sy10 both mutations are expressed simultaneously and independently of each other. A study of joint inheritance of mutations sy1 and sy10 revealed their interaction by means of recessive epistasis: the double mutants has the sy10 phenotype. This means that the sy10 gene controls an earlier stage of synapsis in meiotic prophase than the sy1 gene. Mutation mei8 is inherited independently of sy1 but it is linked to sy10 (recombination frequency 26.8 +/- 3.58%).     

The expression of mutation sy2 causing nonhomologous synapsis in meiosis of diploid rye Secale cereale L

The cytological expression of spontaneous mutation sy2 isolated from a population of weedy rye was examined. It was demonstrated that the primary defect of meiosis in the mutant plants is nonhomologous synapsis, which occurs simultaneously with the homologous one. An electron microscope study of the synaptonemal complex (SC) at prophase I showed synaptic abnormalities that manifested as "switches" of synapting axial elements to the nonhomologous partner and the formation of foldbacks of lateral SC elements. The sy2 mutants are characterized by one to two such events per meiosis. Nonhomologous synapsis leads to the appearance of univalents at metaphase I (on average 4.16 +/- 0.022 per meiocyte) and multivalents (on average 0.12 +/- 0.007 per meiocyte). The presence of multivalents in 12.0% of meiocytes at metaphase I may result from recombination in ectopic regions of homology. It is suggested that the sy2 mutation impairs a component of the system that limits synapsis in meiocytes to only homologous chromosome pairs.     

Genetic control of chromosomal synapsis in Secale cereale L. rye meiosis: sy19 gene, causing heterologous synapsis

Analysis of manifestation and inheritance of a new mutation inducing irregular synapsis in rye showed that abnormal phenotype is determined by a recessive allele of the sy19 gene. In the homozygotes for this mutation, even at the light microscopic level, abnormal formation of bivalents is already observed at pachytene-diakinesis. At metaphase I, the univalent frequency varies from 0 to 14; in a few cells, multivalent associations of chromosomes, which are not clearly oriented in the spindle, are detected. Electron microscopy of synaptonemal complexes revealed both homologous and heterologous synapsis in homozygotes for sy19, namely partial loss of the ability to stringent homology search. Analysis of joint inheritance of sy19 and asynaptic sy1 mutations showed that they are nonallelic, inherited independently, and interact by recessive epistasis. The phenotype of double sy1sy19 mutants indicates that the sy19 gene conditioning heterologous synapsis operates at meiosis later than the synaptic gene sy1. The epistatic group of mutations, sy9 > sy1 > sy19 and sy3, was determined.     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Genetic Analysis of Inheritance ofmei8, sy1, and sy10Meiotic Mutations in Rye Secale cereale L.

It is shown that mutations mei8 (irregular condensation and fragmentation of meiotic chromosomes),sy1(asynapsis), and sy10 (heterologous synapsis) of rye Secale cereale L. are nonallelic. In double mutants mei8 sy1 and mei8 sy10 both mutations are expressed simultaneously and independently of each other. A study of joint inheritance of mutations sy1 and sy10 revealed their interaction by means of recessive epistasis: the double mutants sy1sy10 has the sy10 phenotype. This means that the sy10 gene controls an earlier stage of synapsis in meiotic prophase than the sy1 gene. Mutation mei8is inherited independently of sy1 but it is linked to sy10 (recombination frequency 26.8 ± 3.58%).     

The Expression of Mutation sy2 Causing Nonhomologous Synapsis in Meiosis of Diploid Rye Secale cereale L.

The cytological expression of spontaneous mutation sy2 isolated from a population of weedy rye was examined. It was demonstrated that the primary defect of meiosis in the mutant plants is nonhomologous synapsis, which occurs simultaneously with the homologous one. An electron microscope study of the synaptonemal complex (SC) at prophase I showed synaptic abnormalities that were manifested as switches of synapting axial elements to the nonhomologous partner and the formation of foldbacks of lateral SC elements. The sy2 mutants are characterized by one to two such events per meiocyte. Nonhomologous synapsis leads to the appearance of univalents at metaphase I (on average 4.16 ± 0.002 per meiocyte) and multivalents (on average 0.12 ± 0.007 per meiocyte). The presence of multivalents in 12% of meiocytes at metaphase I may result from recombination in ectopic regions of homology. It is suggested that the sy2 mutation impairs a component of the system that limits synapsis in meiocytes to only homologous chromosome pairs.     

Identification and analysis of DYAD: a gene required for meiotic chromosome organisation and female meiotic progression in Arabidopsis

The dyad mutant of Arabidopsis was previously identified as being defective in female meiosis. We report here the analysis of the DYAD gene. In ovules and anthers DYAD RNA is detected specifically in female and male meiocytes respectively, in premeiotic interphase/meiotic prophase. Analysis of chromosome spreads in female meiocytes showed that in the mutant, chromosomes did not undergo synapsis and formed ten univalents instead of five bivalents. Unlike mutations in AtDMC1 and AtSPO11 which also affect bivalent formation as the univalent chromosomes segregate randomly, the dyad univalents formed an ordered metaphase plate and underwent an equational division. This suggests a requirement for DYAD for chromosome synapsis and centromere configuration in female meiosis. The dyad mutant showed increased and persistent expression of a meiosis-specific marker, pAtDMC1::GUS during female meiosis, indicative of defective meiotic progression. The sequence of the putative protein encoded by DYAD did not reveal strong similarity to other proteins. DYAD is therefore likely to encode a novel protein required for meiotic chromosome organisation and female meiotic progression.     

The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.)

The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene transition but cluster slowly if at all as meiosis proceeds. Intermediate stages in telomere clustering including miniclusters are observed in pam1 but not in wild-type meiocytes. The tight bouquet normally seen at zygotene is a rare event. In contrast, the polarization of centromeres vs. telomeres in the nucleus at the leptotene-zygotene transition is the same in mutant and wild-type cells. Defects in homologous chromosome synapsis include incomplete synapsis, nonhomologous synapsis, and unresolved interlocks. However, the number of RAD51 foci on chromosomes in pam1 is similar to that of wild type. We suggest that the defects in homologous synapsis and the retardation of prophase I arise from the irregularity of telomere clustering and propose that pam1 is involved in the control of bouquet formation and downstream meiotic prophase I events.     

Genetic Control of Chromosome Synapsis at Meiosis in Rye Secale cereale L.: The sy19 Gene Controlling Heterologous Synapsis

Analysis of manifestation and inheritance of a new mutation inducing irregular synapsis in rye showed that abnormal phenotype is determined by a recessive allele of the sy19 gene. In the homozygotes for this mutation, even at the light microscopic level, abnormal formation of bivalents is already observed at pachytene–diakinesis. At metaphase I, the univalent frequency varies from 0 to 14; in a few cells, multivalent associations of chromosomes, which are not clearly oriented in the spindle, are detected. Electron microscopy of synaptonemal complexes revealed both homologous and heterologous synapsis in homozygotes for sy19, namely partial loss of the ability to stringent homology search. Analysis of joint inheritance of sy19 and asynaptic sy1 mutations showed that they are nonallelic, inherited independently, and interact by recessive epistasis. The phenotype of doublesy1sy19 mutants indicates that thesy19 gene conditioning heterologous synapsis operates at meiosis later than the synaptic gene sy1. The epistatic group of mutations, sy9 > sy1 > sy19 and sy3, was determined.     

Meiotic mutants of rye Secale cereale L.

Summary A mutant form of weedy rye characterized by male and female sterility and having a hereditary block in the chromosome synapsis has been found and described. Genetic analysis has shown the synapsis block to be determined by the recessive allele of a gene designated as sy-1. Electron microscopy of surface-spread microsporocyte nuclei revealed the complete absence of the synaptonemal complex over the whole meiotic prophase I, although the axial cores were perfectly formed by each chromosome. Only univalents were observed at metaphase I, their average number ranging from 13.1 to 14.0 per cell. A precocious distribution of univalents at the poles is observed at metaphase I. All of the later stages of meiosis were irregular and resulted in the formation of abnormal microspores. Thus, the mutant proves to be asynaptic because of the blocked initiation of synapses at prophase I.     

Genetic analysis of mutation sy2 which causes nonhomologous meiotic synapsis in chromosomes of diploid rye Secale cereale L

Partially nonhomologous (heterologous) synapsis of meiotic chromosomes in a spontaneous desynaptic mutant form of rye is determined by two recessive genes, sy2a and sy2b, that have independent expression and inheritance. The third gene, dominant inhibitor suppressing the mutant phenotype, has been revealed in hybrid combinations between sy2 mutants and lines segregating other meiotic mutants: sy10 (heterologous synapsis), sy1, and sy9 (asynapsis). All three genes determining desynapsis (sy2a, sy2b, and I) were shown to be nonallelic to monogenic mutations sy10, sy1, and sy9, inherited independently of them and expressed at later stages of prophase I than the sy10 gene. The possibility of modifying monogenic segregation of mutation sy2 by gametophyte selection for a locus linked to the gene expressed as sy2 at particular frequencies of recombination between this gene and selected locus is discussed.     

AtSPO11-1 is necessary for efficient meiotic recombination in plants

The Saccharomyces cerevisiae Spo11 protein catalyses DNA double-strand breaks (DSBs) that initiate meiotic recombination. The model plant Arabidopsis thaliana possesses at least three SPO11 homologues. T-DNA and ethyl-methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11-1 gene is disrupted. Both male and female meiocytes formed very few bivalents. Furthermore, no fully synapsed chromosomes were observed during prophase I. Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non-functional gametes. These meiotic aberrations were associated with a drastic reduction in meiotic recombination. Thus, our data show that initiation of meiotic recombination by SPO11- induced DSBs is a mechanism conserved in plants. Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.     

Abnormal condensation of meiotic chromosomes caused by the mei8 mutation in rye Secale cereale L

Inheritance of two spontaneous meiosis-specific mutations with similar cytologic phenotype was studied. Both mutations were independently obtained from two rye populations (Vyatka variety and weedy rye). Both mutations are recessive, allelic, and monogenically inherited; the corresponding gene is designated mei8. The mutant alleles of the gene cause abnormal meiotic chromosome structure expressed as irregular compaction along the chromosome length, chromatin stickiness at all stages of meiosis, and chromosome fragmentation in anaphase I.     

Expression and inheritance of a desynaptic phenotype with impaired homologous synapsis in rye

The cytological phenotype was studied in a desynaptic form isolated from a population of rye cultivar Vyatka. The primary defect of desynaptic plants was identified as nonhomologous (heterologous) chromosome synapsis, which was observed by electron microscopy of synaptonemal complexes (SCs) in meiotic prophase I. Synapsis defects involved switches of synapsing axial elements to nonhomologous partners, asynapsis in the switching region, and foldbacks formed by the SC lateral elements. Defective bivalent formation was observed at later stages: the univalent number varied and multivalent chromosome associations were observed in single cells in metaphase I. The desynaptic phenotype was controlled by two recessive genes, sy8a and sy8b, which acted and were inherited independently. In a hybrid combination with line Ku-2/63, the desynaptic phenotype was suppressed by the dominant allele of a third gene for inhibitor I; the segregation in hybrid families corresponded to 57:7.     

Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8

A novel mutation, mei8, was isolated in a forward genetic screen for infertility mutations induced by chemical mutagenesis of ES cells. Homozygous mutant mice are sterile. Mutant females exhibit ovarian dysgenesis and lack ovarian follicles at reproductive maturity. Affected males have small testes due to arrest of spermatogenesis during meiotic prophase I. Genetic mapping and positional cloning of mei8 led to the identification of a mutation in Rec8, a homolog of the yeast meiosis-specific cohesin gene REC8. Analysis of meiosis in Rec8(mei8)/Rec8(mei8) spermatocytes showed that, while initiation of recombination and synapsis occurs, REC8 is required for the completion and/or maintenance of synapsis, cohesion of sister chromatids, and the formation of chiasmata, as it is in other organisms. However, unlike yeast and Caenorhabditis elegans, localization of REC8 on meiotic chromosomes is not required for the assembly of axial elements.     

Semisterile meiotic mutant <Emphasis Type="Italic">sy11</Emphasis> with heterologous chromosome synapsis in rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

A study was made of the expression and inheritance of the sy11 mutation, which alters homologous chromosome synapsis in meiotic prophase I of rye. The abnormal phenotype proved to be determined by a recessive allele of a single sy11 gene. Univalents and multivalents were observed in homozygotes for the mutant allele. Analysis of the synaptonemal complex revealed a combination of homologous and nonhomologous synapsis in the mutant. The nonhomologous synapsis frequency significantly decreased in the course of meiotic prophase I in the mutant. The number of chiasmata per bivalent in metaphase I was 1.1 ± 0.01 versus 1.8 ± 0.01 in wild-type plants, and the number of univalents was 2.7 ± 0.06 versus 0.5 ± 0.05 in wild-type plants. As a result, a broad range of abnormalities was observed at subsequent stages of meiosis and led to the formation of defective microspores. Mutant plants were semisterile.     

Molecular assembly of meiotic proteins Asy1 and Zyp1 and pairing promiscuity in rye (Secale cereale L.) and its synaptic mutant sy10

Assembly of two orthologous proteins associated with meiotic chromosome axes in Arabidopsis thaliana (Asy1 and Zyp1) was studied immunologically at meiotic prophase of meiosis of wild-type rye (Secale cereale) and its synaptic mutant sy10, using antibodies derived from A. thaliana. The temporal and spatial expression of the two proteins were similar in wild-type rye, but with one notable difference. Unlike A. thaliana, in which foci of the transverse filament protein Zyp1 appear to linearize commensurately with synapsis, linear tracts of Asy1 and Zyp1 protein form independently at leptotene and early zygotene of rye and coalign into triple structures resembling synaptonemal complexes (SCs) only at later stages of synapsis. The sy10 mutant used in this study also forms spatially separate linear tracts of Asy1 and Zyp1 proteins at leptotene and early zygotene, and these coalign but do not form regular triple structures at midprophase. Electron microscopy of spread axial elements reveals extensive asynapsis with some exchanges of pairing partners. Indiscriminate SCs support nonhomologous chiasma formation at metaphase I, as revealed by multi-color fluorescence in situ hybridization enabling reliable identification of all the chromosomes of the complement. Scrutiny of chiasmate associations of chromosomes at this stage revealed some specificity in the associations of homologous and nonhomologous chromosomes. Inferences about the nature of synapsis in this mutant were drawn from such observations.     

Chromosome pairing in meiosis of partially fertile wheat/rye hybrids

The normal course of meiosis depends on regular pairing of homologous chromosomes. In intergeneric hybrids, including those of wheat, there is no chromosome pairing because there are no homologs. In F₁ wheat/rye hybrids, pairing is largely prevented by the pairing homoeologous1 (Ph1) gene. In its presence, there are only rare instances of pairing; most chromosomes are univalent, and their orientation at metaphase I initiates different pathways of the meiotic cycle. The meiotic-like pathway includes a combination of the reductional and the equational + reductional steps at AI followed by the second division. The resulting gametes are mostly non-functional. The mitotic-like pathway involves equational division of univalents at AI and the absence of the second division. Any fertility of wheat/rye hybrids depends on the production of unreduced gametes arising from meiotic restitution (mitotic-like division). We examined the meiotic pairing in wheat/rye hybrids created from wheat lines with single rye chromosome substitutions and Ph1 present. This guaranteed F₁ meiosis with one pair of rye homologs. All hybrids formed bivalents, but proportions of meiocytes with bivalents varied. In the meiocytes where bivalents were present, there was a higher tendency for the meiotic-like pathway, while in meiocytes where bivalent pairing failed, the tendency was stronger for the mitotic-like pathway. Among the equationally dividing cells, we observed more than 90 % of meiocytes without bivalents, where rye homologs did not form bivalents, too. The data indicate a potential application of wheat/rye lines in producing genetic stocks of amphidiploids with designated genomic constitutions.