Key message Exposure of wheat to high temperatures during male meiosis prevents normal meiotic progression and reduces grain number. We define a temperature-sensitive period and link heat tolerance to chromosome 5D. AbstractThis study assesses the effects of heat on meiotic progression and grain number in hexaploid wheat (Triticum aestivum L. var. Chinese Spring), defines a heat-sensitive stage and evaluates the role of chromosome 5D in heat tolerance. Plants were exposed to high temperatures (30 or 35 °C) in a controlled environment room for 20-h periods during meiosis and the premeiotic interphase just prior to meiosis. Examination of pollen mother cells (PMCs) from immature anthers immediately before and after heat treatment enabled precise identification of the developmental phases being exposed to heat. A temperature-sensitive period was defined, lasting from premeiotic interphase to late leptotene, during which heat can prevent PMCs from progressing through meiosis. PMCs exposed to 35 °C were less likely to progress than those exposed to 30 °C. Grain number per spike was reduced at 30 °C, and reduced even further at 35 °C. Chinese Spring nullisomic 5D-tetrasomic 5B (N5DT5B) plants, which lack chromosome 5D, were more susceptible to heat during premeiosis–leptotene than Chinese Spring plants with the normal (euploid) chromosome complement. The proportion of plants with PMCs progressing through meiosis after heat treatment was lower for N5DT5B plants than for euploids, but the difference was not significant. However, following exposure to 30 °C, in euploid plants grain number was reduced (though not significantly), whereas in N5DT5B plants the reduction was highly significant. After exposure to 35 °C, the reduction in grain number was highly significant for both genotypes. Implications of these findings for the breeding of thermotolerant wheat are discussed.

At the onset of meiosis, chromosomes first decondense and then condense as the process of recognition and intimate pairing occurs between homologous chromosomes. We show here that okadaic acid, a drug known to induce chromosome condensation, can be introduced into wheat interspecific hybrids prior to meiosis to induce chromosome pairing. This pairing occurs in the presence of the Ph1 locus, which usually suppresses pairing of related chromosomes and which we show here delays condensation. Thus the timing of chromosome condensation during the onset of meiosis is an important factor in controlling chromosome pairing.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-010-0185-0) contains supplementary material, which is available to authorized users.

Key messageThe ‘breaker’ element (GcB) of the gametocidal locus derived fromAegilops sharonensishas been mapped to a region proximal to a block of sub-telomeric heterochromatin on chromosome 4SshL.AbstractThe production of alien chromosome addition lines allows the transfer of useful genetic variation into elite wheat varieties from related wild species. However, some wild relatives of wheat, particularly those within the Sitopsis section of the genus Aegilops, possess chromosomes that are transmitted preferentially to the offspring when addition lines are generated. Species within the Sitopsis group possess the S genome, and among these species, Aegilops sharonensis (2n = 14, SshSsh) carries the Ssh genome which is closely related to the D genome of hexaploid wheat. Some S genome chromosomes carry gametocidal loci, which induce severe chromosome breakage in gametes lacking the gametocidal chromosome, and hence, result in gamete abortion. The preferential transmission of gametocidal loci could be exploited in wheat breeding, because linking gametocidal loci with important agronomic traits in elite wheat varieties would ensure retention of these traits through successive generations. In this study, we have mapped the breaker element of the gametocidal locus derived from Ae. sharonensis to the region immediately proximal to a block of sub-telomeric heterochromatin on the long arm of chromosome 4Ssh.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-015-2489-x) contains supplementary material, which is available to authorized users.

Key messageThe meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures. AbstractWe have assessed the effects of high and low temperatures on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (Triticum aestivum L.) variety 'Chinese Spring'. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene (Ltp1) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low (13°C) or high (30°C) temperatures in controlled environment rooms during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after seven days at 13°C. This suggests that the mutant, which we name ttmei1 (temperature tolerance in meiosis 1) has a deletion of a gene that, like Ltp1, normally stabilises chromosome pairing at low temperatures. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ttmei1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. After 24 hours at 30°C, ttmei1 mutants exhibited a reduced number of crossovers and increased univalence, but to a lesser extent than at 13°C. KASP genotyping revealed that ttmei1 has a 4 Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low (and possibly high) temperatures is the meiotic recombination gene Dmc1. KeywordsWheat, high temperature, low temperature, meiosis, chromosome crossover, Dmc1Author contribution statement TD generated the gamma deletion lines, phenotyped the Chinese Spring lines at high and low temperature and identified the ttmei1 deletion mutant. TD and AP carried out the KASP genotyping.M-DR scored chromosome crossover and performed the statistical analysis. AM carried out the immunolocalisation experiments and produced the immunolocalisation figure. AKA identified putative meiosis-specific genes in the 4 Mb deleted region of ttmei1, carried out the sequence 3 alignments and produced the gene expression figures; TD produced all other figures and wrote the manuscript. PS provided a critique of the approach and contributed to discussion of the results. GM provided the concept, provided thoughts and guidance and revised and edited the manuscript. Conflict of interestThe authors declare that they have no conflict of interest. 5DL-9 and 5DL-13 (Endo and Gill 1996). DNA extractionsPlants were initially grown in modular trays in a controlled environment room (CER) at 20°C (day) and 15°C (night) with a 16-hour photoperiod (lights on between 10:00 and 02:00) and 70 % humidity.Wheat seedlings were grown to the ...

Key message The meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures. Abstract We initially assessed the effects of low temperature on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (Triticum aestivum L.) variety 'Chinese Spring'. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene (Ltp1) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low temperature in a controlled environment room during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after 7 days at 13 °C, suggesting that Ltp1 is deleted in this mutant. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ltp1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. KASP genotyping revealed that the ltp1 mutant has a 4-Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low temperatures is the meiotic recombination gene Dmc1. The ltp1 mutants were subsequently treated at 30 °C for 24 h during meiosis and exhibited a reduced number of crossovers and increased univalence, though to a lesser extent than at 13 °C. We therefore renamed our ltp1 mutant 'ttmei1' (temperature-tolerant meiosis 1) to reflect this additional loss of high temperature tolerance.

Tetraploid and hexaploid wheat have multiple genomes, with successful meiosis and preservation of fertility relying on synapsis and crossover only taking place between homologous chromosomes. In hexaploid wheat, the major meiotic geneTaZIP4-B2(Ph1) on chromosome 5B, promotes crossover between homologous chromosomes, whilst suppressing crossover between homeologous (related) chromosomes. Tetraploid wheat has threeZIP4copies:TtZIP4-A1on chromosome 3A,TtZIP4-B1on 3B andTtZIP4-B2on 5B. Previous studies showed thatZIP4mutations eliminate approximately 85% of crossovers, consistent with loss of the class I crossover pathway. Here, we show that disruption of twoZIP4gene copies inTtzip4-A1B1double mutants, results in a 76-78% reduction in crossovers when compared to wild-type plants. Moreover, when all three copies are disrupted inTtzip4-A1B1B2triple mutants, crossover is reduced by over 95%, suggesting that theTtZIP4-B2copy is also affecting class II crossovers. This implies that, in wheat, the class I and class II crossover pathways may be interlinked. WhenZIP4duplicated and diverged from chromosome 3B on wheat polyploidization, the new 5B copy,TaZIP4-B2, may have acquired an additional function to stabilize both crossover pathways. In plants deficient in all threeZIP4copies, synapsis is delayed and does not complete, consistent with our previous studies in hexaploid wheat, when a similar delay in synapsis was observed in a 59.3Mb deletion mutant,ph1b, encompassing theTaZIP4-B2gene on chromosome 5B. These findings confirm the requirement ofZIP4-B2for efficient synapsis, and suggest thatTtZIP4genes have a stronger effect on synapsis than previously described in Arabidopsis and rice. Thus,ZIP4-B2accounts for the two major phenotypes reported forPh1, promotion of homologous synapsis and suppression of homeologous crossover.

Effective chromosome synapsis and crossover formation during meiosis are essential for fertility, especially in grain crops such as wheat. These processes function most efficiently in wheat at temperatures between 17-23 °C, although the genetic mechanisms for such temperature dependence are unknown. In a previously identified mutant of the hexaploid wheat reference variety ‘Chinese Spring’ lacking the long arm of chromosome 5D, exposure to low temperatures during meiosis resulted in asynapsis and crossover failure. In a second mutant (ttmei1), containing a 4 Mb deletion in chromosome 5DL, exposure to 13 °C led to similarly high levels of asynapsis and univalence. Moreover, exposure to 30 °C led to a significant, but less extreme effect on crossovers. Previously, we proposed that, of 41 genes deleted in this 4 Mb region, the major meiotic gene TaDMC1-D1 was the most likely candidate for preservation of synapsis and crossovers at low (and possibly high) temperatures. In the current study, using RNA-guided Cas9, we developed a new Chinese Spring CRISPR mutant, containing a 39 bp deletion in the 5D copy of DMC1, representing the first reported CRISPR-Cas9 targeted mutagenesis in Chinese Spring, and the first CRISPR mutant for DMC1 in wheat. In controlled environment experiments, wild-type Chinese Spring, CRISPR dmc1-D1 and backcrossed ttmei1 mutants were exposed to either high or low temperatures during the temperature-sensitive period from premeiotic interphase to early meiosis I. After 6-7 days at 13 °C, crossovers decreased by over 95% in the dmc1-D1 mutants, when compared with wild-type plants grown under the same conditions. After 24 hours at 30 °C, dmc1-D1 mutants exhibited a reduced number of crossovers and increased univalence, although these differences were less marked than at 13 °C. Similar results were obtained for ttmei1 mutants, although their scores were more variable, possibly reflecting higher levels of background mutation. These experiments confirm our previous hypothesis that DMC1-D1 is responsible for preservation of normal crossover formation at low and, to a certain extent, high temperatures. Given that reductions in crossovers have significant effects on grain yield, these results have important implications for wheat breeding, particularly in the face of climate change.

Effective chromosome synapsis and crossover during meiosis are essential for fertility, especially in grain crops such as wheat. These processes function most efficiently in wheat at temperatures between 17-23 °C, although the genetic mechanisms for such temperature dependence are unknown. In a previously identified mutant of the hexaploid wheat reference variety 'Chinese Spring' lacking the long arm of chromosome 5D, exposure to low temperatures during meiosis resulted in asynapsis and crossover failure. In a second mutant (ttmei1), containing a 4 Mb deletion in chromosome 5DL, exposure to 13 °C led to similarly high levels of asynapsis and univalence. Moreover, exposure to 30 °C led to a significant, but less extreme effect on crossover. Previously, we proposed that, of 41 genes deleted in this 4 Mb region, the major meiotic geneTaDMC1-D1was the most likely candidate for preservation of synapsis and crossover at low (and possibly high) temperatures. In the current study, using RNA-guided Cas9, we developed a new Chinese Spring CRISPR mutant, containing a 39 bp deletion in the 5D copy ofDMC1, representing the first reported CRISPR-Cas9 targeted mutagenesis in Chinese Spring, and the first CRISPR mutant forDMC1in wheat. In controlled environment experiments, wild-type Chinese Spring, CRISPRdmc1-D1and backcrossedttmei1mutants were exposed to either high or low temperatures during the temperature-sensitive period from premeiotic interphase to early meiosis I. After 6-7 days at 13 °C, crossover decreased by over 95% in thedmc1-D1mutants, when compared with wild-type plants grown under the same conditions. After 24 hours at 30 °C,dmc1-D1mutants exhibited a reduced number of crossovers and increased univalence, although these differences were less marked than at 13 °C. Similar results were obtained forttmei1mutants, although their scores were more variable, possibly reflecting higher levels of background mutation. These experiments confirm our previous hypothesis thatDMC1-D1is responsible for preservation of normal synapsis and crossover at low and, to a certain extent, high temperatures. Given that reductions in crossover have significant effects on grain yield, these results have important implications for wheat breeding, particularly in the face of climate change.