The plant material used in this study and its production is described in Figure 1, and includes: hexaploid wheat Triticum aestivum cv. Chinese Spring (2n = 6× = 42; AABBDD), either containing or lacking the Ph1 locus (Sears, 1977); rye Secale cereale cv Petkus (2n = 2× = 14; RR); wheat–rye hybrids crosses between hexaploid wheat either containing or lacking the Ph1 locus, and rye; octoploid triticale × Triticosecale Wittmack (2n = 8× = 56), obtained after genome duplication of wheat–rye hybrids either containing or lacking the Ph1 locus.

The wheat–rye hybrids were generated by either crossing T. aestivum cv. Chinese Spring or T. aestivum cv. Chinese Spring ph1b mutant (Sears, 1977) as the female parent, with S. cereale cv. Petkus. Interspecific wheat–rye hybrids, either containing or lacking the Ph1 locus, are completely sterile. The octoploid triticales were generated by treating wheat–rye hybrids, either containing or lacking the Ph1 locus, with colchicine to double the chromosome number. Colchicine was applied according to the capping technique (Bell, 1950). Briefly, when the hybrids were at the 4–5 tillering stage, two of the tillers were cut and covered (or capped) with a small glass phial containing 0.5 ml of a solution of 0.3% colchicine. Once the solution was absorbed by the plant, the hybrids were left to grow. Successful chromosome doubling results in seed set. A few of the resulting duplicated seeds obtained were selfed twice in order to have sufficient seeds for all the studies. Only plants with a euploid chromosome number of 56 chromosomes were used for RNA-seq sample collection. One spike of every plant used for the RNA-seq analysis was selfed, so that cytological analysis could be performed on the progeny. One of the triticales lacking Ph1 used for the RNA-seq was sterile, so instead, another triticale lacking Ph1 was used to complete the cytological analysis.

Seeds were germinated on Petri dishes for 3 to 4 days. The seedlings were vernalized for 3 weeks at 7°C and then transferred to a controlled environment room until meiosis, under the following growth conditions: 16 h light/8 h night photoperiod at 20°C day and 15°C night, with 70% humidity. After 6 to 7 weeks, plants were ready for meiosis studies. Tillers were harvested after 6 to 7 weeks, at early booting stage, when the flag leaf ligule is just visible (39 Zadoks scale). Anthers were collected at early prophase, at the transition leptotene-zygote, which is during the telomere bouquet stage in wheat. For each dissected floret, one of the three synchronized anthers was squashed in 45% acetic acid in water to identify the meiotic specific stage. The two remaining anthers were harvested into RNAlater (Ambion, Austin, TX, United States) for the RNA-seq experiments or fixed in 100% ethanol/acetic acid 3:1 (v/v) for cytological analysis of meiocytes.

Anthers from wheat, wheat–rye hybrids and triticale, both in the presence and absence of the Ph1 locus, and rye were collected as described in the “Plant Material” section. Three biological replicates were prepared for each genotype, so a total of 21 samples were obtained. Anthers at the selected meiotic stage were harvested into RNAlater (Ambion, Austin, TX, United States). The anthers from three plants of each genotype were pooled in a 1.5-ml Eppendorf tube until 300 anthers were collected. As there were so few triticale seeds available, each triticale sample was derived from a single plant. Once sufficient anthers had been collected, the material was squashed using a pestle to release the meiocytes from the anthers, and the mix was transferred to a new Eppendorf trying to avoid as much of the anther debris as possible, to enrich the sample with meiocytes. The enriched meiocyte samples were centrifuged to eliminate the RNAlater and homogenized using QIAshredder spin columns (Qiagen, Hilden, Germany). RNA extraction was performed using a miRNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. This protocol allows purification of a separate miRNA-enriched fraction (used for further analysis) and the total RNA fraction (>200 nt) used in this study.

One microgram of RNA was purified to extract mRNA with a poly-A pull down using biotin beads. A total of 21 libraries were constructed using the NEXTflex^TM Rapid Directional RNA-Seq Kit (Bioo Scientific Corporation, Austin, TX, United States) with the NEXTflex^TM DNA Barcodes–48 (Bioo Scientific Corporation, Austin, TX, United States) diluted to 6 μM. The library preparation involved an initial QC of the RNA using Qubit DNA (Life Technologies, Carlsbad, CA, United States) and RNA (Life Technologies, Carlsbad, CA, United States) assays, as well as a quality check using the PerkinElmer GX with the RNA assay (PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA, United States). The constructed stranded RNA libraries were normalized and equimolar pooled into one final pool of 5.5 nM using elution buffer (Qiagen, Hilden, Germany). The library pool was diluted to 2 nM with NaOH, and 5 μl was transferred into 995 μl HT1 (Illumina) to give a final concentration of 10 pM. Diluted library pool of 120 μl was then transferred into a 200-μl strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot. The flow cell was clustered using HiSeq PE Cluster Kit v4, utilizing the Illumina PE_HiSeq_Cluster_Kit_V4_cBot_recipe_V9.0 method on the Illumina cBot. Following the clustering procedure, the flow cell was loaded onto the Illumina HiSeq 2500 instrument following the manufacturer’s instructions. The sequencing chemistry used was HiSeq SBS Kit v4 with HiSeq Control Software 2.2.58 and RTA 1.18.64. The library pool was run in a single lane for 125 cycles of each paired end read. Reads in bcl format were demultiplexed based on the 6 bp Illumina index by CASAVA 1.8, allowing for a one base-pair mismatch per library and converted to FASTQ format by bcl2fastq. RNA-seq data processing: the raw reads were processed using SortMeRNA v2.0 (Kopylova et al., 2012) to remove rRNA reads. The non-rRNA reads were then trimmed using Trim Galore v0.4.1¹ to remove adaptor sequences and low-quality reads (-q 20 – length 80 – stringency 3).

RNA-seq reads (trimmed non-rRNA reads as described above) for the 18 samples were aligned to the RefSeqv1.0 assembly (International Wheat Genome Sequencing Consortium [IWGSC], 2018), using HISAT v2.0.5 with strict mapping options (–no-discordant –no-mixed -k 1 –phred33 –rna-strandness RF) to reduce noise caused by reads mapping to the wrong regions. Output bam files (binary format for storing sequence alignment data) were sorted using samtools v1.5. The normalized average chromosome coverage (depth of reads aligning to each base along the genome) per 1 million base windows was obtained using bedtools v2.24.0. GenomeCoverageBed was run to generate a bedgraph file containing base coverage along each chromosome (scaled by reads per million). Each of the 21 chromosomes were divided into 1 million base windows and bedtools map was run to compute the average read depth over each 1 million base window. The average coverage was obtained for the three biological replicates of each sample. The ratio of coverage between samples was plotted as a heatmap using a custom R script². The following formula was used to calculate the ratio (-1 to +1) of coverage; ratio = sample1–sample2/sample1+sample2.

Genes were examined for differential expression between wheat containing and wheat lacking the Ph1 locus, by pseudoaligning the raw reads for these six samples against the Chinese Spring RefSeqv1.0+UTR transcriptome reference (International Wheat Genome Sequencing Consortium [IWGSC], 2018), using Kallisto v 0.42.3 (Bray et al., 2016) with default options. The index was built using a k-mer length of 31. Transcript abundance was obtained as estimated counts and transcripts per million (TPM) for each sample, and all samples were merged into matrices of gene-level expression using the script merge_kallisto_output_per_experiment_with_summary.rb from expVIP³. Only genes with a mean expression >0.5 TPM in at least one condition, i.e., one of the genotypes (wheat containing or wheat lacking Ph1) were retained for differential expression analysis (Ramírez-González et al., 2018); this included 65,683 genes. Differential expression analysis between conditions (three replicates for each condition) was carried out using DESeq2 (v1.18.1) in R (v3.4.4). The numbers of differentially expressed genes at various thresholds (padj < 0.05, padj < 0.01, and padj < 0.001, and fold change > 2) were obtained. Gene Ontology (GO) enrichment was carried out using the R package goseq (v1.26.0) in R (v3.4.4). The GO term annotation was obtained from the RefSeqv1.0 (International Wheat Genome Sequencing Consortium [IWGSC], 2018). Genes up-regulated >twofold with padj < 0.01, and genes down-regulated >twofold with padj < 0.01 were analyzed separately. Only significantly enriched GO terms (padj < 0.05) were retained. Human readable, PFAM and InterPro functional annotations for individual differentially expressed genes of interest were obtained from the functional annotation of the RefSeqv1.0 (International Wheat Genome Sequencing Consortium [IWGSC], 2018) from https://opendata.earlham.ac.uk/wheat/under_license/toronto/Ramirez-Gonzalez_etal_2018-06025-Transcriptome-Landscape/data/TablesForExploration/FunctionalAnnotation.rds (Ramírez-González et al., 2018).

Differential expression amongst wheat–rye hybrid and triticale samples was analyzed by constructing an in silico wheat+rye transcriptome through combining the Chinese Spring RefSeqv1.0+UTR transcriptome reference (International Wheat Genome Sequencing Consortium [IWGSC], 2018) with the published rye transcriptome (Bauer et al., 2016). In total, this combined reference contained 326,851 transcripts, of which 299,067 were from wheat and 27,784 were from rye. The rye transcriptome only contained one isoform per gene whereas the wheat transcriptome contained multiple isoforms. The 12 samples with both wheat and rye genomes (wheat–rye hybrid and triticale in the presence and absence of Ph1, each with three biological replicates) were mapped to the in silico wheat–rye transcriptome using the same method as described above for wheat samples. Only genes with a mean expression >0.5 TPM in at least one condition (wheat–rye hybrid or triticale, containing or lacking Ph1) were retained for differential expression analysis. This included 83,202 genes in total, of which 50,292 were high confidence (HC) wheat genes, 22,138 were low confidence (LC) wheat genes and 10,772 were rye genes. Two comparisons were carried out to examine the effect of Ph1 (wheat–rye hybrids carrying vs. lacking Ph1, and triticale carrying vs. lacking Ph1), and one comparison performed to examine the effect of different synapsis levels and chromosome doubling (wheat–rye hybrids vs. triticale, both carrying Ph1). Differentially expressed genes for each comparison were identified using DESeq2 as described above, using three replicates per condition. The numbers of differentially expressed genes at various thresholds (padj < 0.05, padj < 0.01, and padj < 0.001, and fold change >2) were obtained for wheat and rye genes. GO term annotation was available for the wheat genes, therefore we focused only on differentially expressed wheat genes for GO enrichment analysis and excluded differentially expressed rye genes from this analysis. GO enrichment analysis was carried out as described for the wheat samples, again separately analyzing genes up-regulated >twofold with padj < 0.01, and genes down-regulated >twofold with padj < 0.01.

The rye genome sequence is still incomplete; therefore, the present study focused on wheat-specific transcription effects. However, the rye data (PRJEB25586) are deposited to facilitate future analyses by the community.

It was not possible to analyze the samples used for the RNA-seq analysis, so their progeny were analyzed instead. One spike of every plant used for the transcription analysis was selfed, and three individuals from each progeny analyzed. One of the triticale lacking Ph1 used for the RNA-seq was sterile, so instead, another triticale lacking Ph1 which had 57 chromosomes, was used.

The preparation of mitotic metaphase spreads and subsequent genomic in situ hybridization (GISH) was carried out as described previously (Rey et al., 2018b). Meiotic metaphase I spread preparation and subsequent GISH were also carried out as described previously (Cabrera et al., 2002). S. cereale, Triticum urartu, and Aegilops tauschii were used as probes to label rye, wheat A- and wheat D-genomes, respectively. S. cereale genomic DNA was labeled with tetramethyl-rhodamine-5-dUTP (Sigma) by nick translation as described previously (Cabrera et al., 2002). T. urartu and Ae. tauschii genomic DNA were labeled with biotin-16-dUTP and digoxigenin-11-dUTP, using the Biotin-nick translation mix and the DIG-nick translation mix, respectively (Sigma, St. Louis, MO, United States) according to the manufacturer’s instructions. Biotin-labeled probes were detected with Streptavidin-Cy5 (Thermo Fisher Scientific, Waltham, MA, United States). Digoxigenin-labeled probes were detected with anti-digoxigenin-fluorescein Fab fragments (Sigma).

Images were acquired using a Leica DM5500B microscope equipped with a Hamamatsu ORCA-FLASH4.0 camera and controlled by Leica LAS X software v2.0. Images were processed using Fiji (an implementation of ImageJ, a public domain program by W. Rasband available from https://imagej.nih.gov/ij/) and Adobe Photoshop CS4 (Adobe Systems Incorporated, United States) version 11.0 × 64.

Raw Illumina reads have been deposited into EMBL-EBI ENA (European Nucleotide Archive⁴) under project number PRJEB25586. Analyzed data for the wheat samples (TPM and counts) were integrated in the expVIP platform www.wheat-expression.com (Borrill et al., 2016). Analyzed data for wheat, hybrids and triticale samples are available through https://opendata.earlham.ac.uk/.

To assess whether synapsis, ploidy level and changes in chromatin structure associated with Ph1 have any effect on global transcription during early meiotic prophase I, the transcriptome of wheat, wheat–rye hybrid and the corresponding triticale were analyzed by RNA-seq in the presence and absence of the Ph1 locus. In wheat florets, the three anthers and the meiocytes within them are highly synchronized in development. We staged one of the three anthers by microscopy, to ensure that the meiocytes were at the transition leptotene-zygotene stage, leaving the other two anthers for RNA extraction. Three biological replicates were produced for each transcriptome, with a total of 18 libraries generated.

Using Illumina sequencing, a total of 1,388 million reads were generated for the 18 libraries. For subsequent analysis, the RNA-seq data were processed using two different methods. Firstly, samples were trimmed and aligned to the wheat RefSeqv1.0 assembly using HISAT to generate the chromosome coverage plots. Strict mapping options were used to reduce the noise caused by reads mapping to the wrong regions, particularly mis-mapping from the rye onto the wheat genome. The percentage of reads aligned to the wheat genome was on average 88.46% for wheat samples, 74.61% for wheat–rye hybrids samples and 75.48% for triticale samples (Supplementary Table S1). On average 13.4% fewer reads mapped in wheat–rye and triticale samples than in wheat samples, indicating that the stringent mapping conditions were effective in reducing mis-mapping. However, it is possible that a low level of residual mis-mapping occurred, whereby reads from rye genes are mapped onto the wheat genome.

Secondly, DESesq2 was used to examine genes differentially expressed between genotypes. The six wheat samples were pseudoaligned to the Chinese Spring RefSeqv1.0+UTR transcriptome reference using kallisto. The percentage of reads pseudoaligned was similar across samples, with a mean value of 72% as detailed in Supplementary Table S2. The three biological replicates showed good correlation and clustered together in the principle component analysis, although samples containing the Ph1 locus grouped together more tightly than samples lacking Ph1 (Supplementary Figure S1A). The wheat RefSeq Annotation v1.0 includes 110,790 HC genes and 158,793 LC genes. LC genes represent partially supported gene models, gene fragments and orphan genes (International Wheat Genome Sequencing Consortium [IWGSC], 2018). We decided to retain these LC genes in our differential expression analysis because one of the reasons for them to be considered as LC genes is a lack of RNA-seq data evidence. As already mentioned, no data on wheat meiosis were previously published, and since it is therefore possible that some of these LC genes are specifically involved in meiosis, we decided to include them in the analysis.

The 12 samples from wheat–rye hybrids and triticale, were mapped to a wheat+rye transcriptome created in silico for this study (described in section “Materials and Methods”). Although our aim in these samples was principally to study the expression of wheat genes, the use of a hybrid transcriptome reduced the possibility of mis-mapping between rye and wheat reads, which would have led to inaccurate quantification of expression. Kallisto was used for mapping because in wheat, it accurately distinguishes reads from homoeologs carrying genes with a sequence identity between 95–97% (Ramírez-González et al., 2018). Therefore, kallisto is capable of distinguishing wheat and rye reads, which are more divergent [91% sequence identity within genes (Khalil et al., 2015)]. The percentage of reads pseudoaligned to this transcriptome was similar across samples, with a mean value of 71% for wheat–rye hybrids and triticale (Supplementary Table S2). The three biological replicates from each genotype showed good correlation and clustered together in the principle component analysis (Supplementary Figure S1B). In total, 269,583 genes from wheat (110,790 HC and 158,793 LC), plus 27,784 genes from rye were annotated, giving a total of 297,367 genes for the hybrid transcriptome created.

Chromosome coverage plots were generated to reveal a global picture of the difference in transcription between the different genotypes analyzed. The cleaned RNA-seq reads were aligned to the RefSeqv1.0 assembly and the ratio of the coverage along all the chromosomes plotted as a heatmap (Figure 2).

At the leptotene-zygotene transition, when the telomere bouquet is tightly formed, only homologous chromosomes can synapse. In wheat–rye hybrids there are no homologs present, and therefore, no synapsis takes place at this stage; whereas in triticale, a significant level of synapsis occurs. However, no overall change in wheat transcription was observed when these two genotypes were compared (Figure 2A and Supplementary Figure S2). Even more striking was that the duplication of the genome and change in ploidy level had little effect on overall wheat transcription. The wheat–rye hybrid is a poly-haploid (n = 4× = 28, ABDR) and triticale an octoploid (2n = 8× = 56, AABBDDRR), and although vegetative development is normal in both genotypes, the haploid hybrids are completely sterile. The reason for sterility in the wheat–rye hybrid is that meiosis is highly compromised, with only one CO at metaphase I and subsequent random segregation of chromosomes. Despite this, wheat transcription during early meiotic prophase seems not to be affected.

Next, a comparison of wheat, wheat–rye hybrid and triticale, with their corresponding genotypes lacking the Ph1 locus was made (Figures 2B–D and Supplementary Figure S2). This time, the heatmaps revealed a very different situation, with very clear differences in transcription in all comparisons. A deletion on chromosome 5B (visualized in dark blue in Figures 2B–D) was observed in all samples lacking Ph1, corresponding to the deletion of the ph1b mutant (Sears, 1977). However, several other deletions (visualized in dark blue) were also observed in all samples. Due to the nature of this locus, which affects synapsis and CO formation between homoeologs, the presence of chromosome rearrangements has been previously described in wheat lacking Ph1 (Sánchez-Morán et al., 2001). However, the number of rearrangements revealed was higher than expected. An interstitial deletion on 3BL and two deletions on 3AL, one interstitial and another terminal, were common to all samples lacking Ph1. Apart from these, wheat lacking Ph1 had two more deletions: a terminal one on 2DL and a distal one on 3DL; while triticale lacking Ph1 had three more deletions: a terminal one on 1AL, a large terminal one on 3AL and a large distal one on 5DL. As observed in Figure 2C, the heatmap belonging to the wheat–rye hybrids showed no difference in overall transcription whether Ph1 was present or absent, apart from the common deletions mentioned. However, heatmaps corresponding to wheat and triticale were more difficult to interpret, with several chromosome regions showing clear differential expression without a completely clear-cut presence or absence of Ph1, as in the case of the deletions. The triticale heatmap (Figure 2D) for example, revealed three chromosome regions showing increased transcription in triticale lacking Ph1 (visualized in orange in the heatmap): a terminal region in 1DL and 3DL, and a large distal region on 5BL. Interestingly, every one of these chromosome regions corresponded to a chromosome deletion on a homoeologous chromosome. For example, the terminal deletion on 1AL, corresponded to the terminal increased transcription on 1DL. Recombination could occur between 1A and 1D in the absence of Ph1, resulting in two 1D chromosomes plus two 1A chromosomes being detected in this material, in which the distal part of 1AL had a chromosome segment from 1DL. This, therefore, resulted in four copies of the D-genome chromosome segment, and hence increased transcription. These observations are consistent with wheat nulli-tetrasomic line analysis (Borrill et al., 2016), where the presence of four copies of a homoeolog leads to a doubling of transcription. The observed increases in transcription associated with the other deletions in wheat and triticale lacking Ph1 could also be explained in a similar manner. There were some regions where the interpretation was more complex. To investigate this further, we created heatmaps of wheat vs. wheat lacking Ph1, and triticale vs. triticale lacking Ph1, for every individual ph1 mutant sample (Supplementary Figures S3, S4). Results revealed that every individual sample lacking Ph1 was different, apart from the rearrangements common to all samples lacking Ph1 described above. One triticale sample was extremely rearranged (Supplementary Figure S4), so we decided to explore this further and perform GISH experiments on this material, which will be described in the following sections.

In summary, at this stage of meiosis, overall transcription was not affected by the absence of the Ph1 locus. Therefore, chromatin changes associated with the Ph1 locus did not affect overall transcription. All significant transcriptional changes observed between genotypes with and without Ph1 were associated with the presence/absence of chromosome regions likely to be the result of homoeologous recombination. We therefore conclude that neither synapsis, level of ploidy nor the presence of Ph1 have a significant overall effect on wheat meiotic transcription.

Chromosome coverage plots showed no global changes in transcription. However, we also wanted to identify the number of genes differentially expressed, and check whether they were related to meiotic processes. A Kallisto-DESeq2 pipeline was used to examine the DEG between genotypes. Only genes expressed >0.5 TPM in at least one of the genotypes were selected for differential expression analysis, the rest being filtered out as non-expressed genes. The number of DEG among samples was calculated using different thresholds as described in Materials and Methods and Supplementary Table S3, with further analysis focussed on the comparisons at padj < 0.01 and fold change >2.

In 1977 (Sears, 1977), the ph1b deletion used in this study (and most studies involving this locus) was obtained and estimated to be of 70 Mb in size. Using our gene expression data and the RefSeqv1.0 assembly, the ph1b deletion is now defined to a 59.3 Mb region containing 1187 genes, from which 299 genes are expressed in our RNA-seq data. The locus was further defined to a smaller region (Griffiths et al., 2006; Al-Kaff et al., 2008), now defined to 0.5 Mb in size and containing 25 genes (7 HC + 18 LC genes), from which only two are expressed in our RNA-seq data. One of these two genes is a DUF2431 domain protein (TraesCS5B01G254900) of unknown function. The other gene is the duplicated ZIP4 gene (TaZIP4-B2), which has been recently identified as the gene responsible for both promoting homologous CO and restricting homoeologous CO (Rey et al., 2017, 2018a). Another gene within the ph1b deletion, termed by the authors as C-Ph1, was also recently proposed to contribute to the Ph1 effect on recombination, specifically during metaphase I (Bhullar et al., 2014); however, this gene does not show any expression in our RNA-seq data. The authors reported 3 copies of this gene, one on 5A (truncated), one on 5B (with a splice variant named 5B^alt) and one on 5D, claiming that only the 5B copy was metaphase I-specific and therefore, responsible for the phenotype characteristic of Ph1. However, blasting these gene sequences against the RefSeqv1.0 assembly showed that 5B^alt was a fourth gene copy located on 5A chromosome just upstream of the original 5A copy (5A-1: TraesCS5A01G381600LC and 5A-2: TraesCS5A01G381700LC). The RNA-seq data obtained in the present study, as well as 849 wheat RNA-seq samples now publicly available (Ramírez-González et al., 2018) and 8 wheat meiotic libraries available at https://urgi.versailles.inra.fr/files/RNASeqWheat/Meiosis/, can now be used to study the expression profile of all the different copies of the ZIP4 and C-Ph1 genes found across a diverse range of tissues and developmental stages (Figure 4). The TaZIP4 copy on 5B (TaZIP4-B2) is the dominant ZIP4 copy and it is expressed in all tissue types, including all meiotic stages. C-Ph1 has an almost tissue-specific expression pattern, limited to stamen tissue during the heading stage (post-meiosis stage), and with the 5D copy being expressed dominantly over all other gene copies (expression level of the 5D copy being >1700 TPM, and of the 5B copy being <31 TPM). None of the C-Ph1 copies is expressed during any meiosis stage, except for the 5D copy that is expressed at a very low level (<4 TPM) in comparison with its expression in anthers at heading (>1700 TPM). In summary, we can confirm that C-Ph1 on 5B is not expressed during meiosis, and cannot therefore be responsible for any Ph1 effect during metaphase I.

Cytological analysis of wheat and wheat–rye hybrids, both in the presence and absence of the Ph1 locus, has been well documented in previous studies (Orellana, 1985; Naranjo et al., 1988; Wang and Holm, 1988; Benavente et al., 1998; Mikhailova et al., 1998; Sánchez-Morán et al., 1999, 2001); however, this is the first time to our knowledge, that triticale lines lacking Ph1 have been generated. As the chromosome coverage plots suggest the presence of several chromosome rearrangements, we performed mitotic and meiotic analysis to explore the origin and extent of these reorganizations.

During meiosis, homologous (and sometimes non-homologous) chromosomes pair and then synapse through the polymerization of a protein structure known as the synaptonemal complex, which provides the structural framework for recombination to take place. Throughout the whole of meiosis, but particularly during the synaptic process, there are multiple changes in chromatin structure and organization (and even positioning in the nucleus), taking place within a relatively short period of time and needing to be highly regulated. In wheat, synapsis is initiated during the telomere bouquet stage at early prophase, during the transition leptotene-zygotene (Martín et al., 2017). Moreover, it has been observed, that the process of recognition between homologs is associated with major changes in the chromatin structure of chromosomes (Prieto et al., 2004), suggesting that these changes in chromatin structure may be required for the homolog recognition process and initiation of synapsis. Indeed, it is now well understood that chromatin conformation is a critical factor in enabling many regulatory elements to perform their biological activity, and that chromatin structure profoundly influences gene expression (Dixon et al., 2015; Doğan and Liu, 2018). Therefore, it was reasonable to suppose that differences in synapsis, and therefore, in chromatin structure would translate into differences in transcription.

Surprisingly, however, we did not find the expected differences in overall wheat transcription and gene expression when comparing samples with different levels of synapsis, indicating that the structural changes associated with this process were not directly coupled to transcription. Probably the clearest example of this was the comparison of wheat–rye hybrids and octoploid triticale. Wheat–rye hybrids possess a haploid set of wheat and rye chromosomes (there are no homologs present) and no synapsis is observed during the telomere bouquet stage (Martín et al., 2017). In contrast, octoploid triticale, which is obtained after chromosome doubling of wheat–rye hybrids, and which therefore possesses a whole set of wheat and rye chromosomes, exhibits extensive synapsis during the same stage. However, only 0.47% of the expressed genes were differentially expressed between these two samples (0.38% considering only HC genes), with most of these genes being involved in stress response and other metabolic processes (general functions) not related to meiosis.

Even more striking is the fact that in our study, global gene expression was not affected by WGD. Several previous studies have reported genetic and epigenomic processes being disrupted after hybridization and polyploidization, with subsequent changes in gene expression (Qi et al., 2012; Renny-Byfield and Wendel, 2014; Khalil et al., 2015; Edger et al., 2017 and references therein; Sun et al., 2017). These studies were not performed on meiotic tissue, however, given the known failure of meiosis in wheat–rye hybrids and the relatively normal meiotic progression in the duplicated triticale, it would have been reasonable to expect an effect on the expression pattern. However, as mentioned above, only a small fraction of genes were differentially expressed, again with none involved in meiosis. Unfortunately, the rye genome sequence is incomplete, and a similar analysis for rye genes could not be performed. In the future, when the complete rye genome sequence is available, it will be interesting to assess whether global expression of rye genes is also unchanged.

We propose that a possible explanation for the striking robustness in gene expression during early meiotic prophase is that the transcription of genes required for the meiotic program has already occurred prior to the leptotene-zygotene transition. Meiosis is a very complex process which takes place in a relatively short period of time. In wheat, the whole meiosis process lasts only 24 h at 20°C, with the whole process of synapsis being less than 6 h long (Bennet et al., 1973). Therefore, it would be reasonable to suppose that most of the transcription needed for such a critical process has already occurred prior to synapsis initiation. From mouse studies, it has been recently reported that a considerable number of genes involved in early, as well as later meiotic processes, are already active at early meiotic prophase (da Cruz et al., 2016). Moreover, a major change in gene expression patterns occurs during the middle of meiotic prophase (pachytene), when most genes related to spermiogenesis and sperm function appear already active (da Cruz et al., 2016). It is possible that this change in gene expression pattern also happens in wheat, with a transcriptional switch from pre-meiosis to meiosis taking place very early, before meiotic prophase. Only when more RNA-seq datasets are available, can the dynamics of gene expression during wheat meiosis be fully understood.

The Ph1 locus in wheat is by far, the best characterized locus involved in the diploid-like behavior of polyploids during meiosis. Ph1 has a dual effect during meiosis: firstly, improving the efficiency of homologous synapsis and secondly, preventing CO formation between homoeologs while increasing CO between homologs (Martín et al., 2017). Recently, the duplicated ZIP4 gene inside the Ph1 locus on 5B (TaZIP4-B2) has been identified as responsible for the effect of this locus on recombination and suggested to be also involved in the improved synapsis efficiency (Rey et al., 2017, 2018a). ZIP4 is a meiotic gene shown to have a major effect on homologous COs in both Arabidopsis and rice, and a mild effect on synapsis (Chelysheva et al., 2007; Shen et al., 2012). Although its exact mode of action is unknown, it seems to act as a hub, facilitating physical interactions between components of the chromosome axis and the CO machinery (Perry et al., 2005; Tsubouchi et al., 2006). In diploid species, knockouts of this gene result in sterility, as failure of homologous COs at metaphase I leads to incorrect segregation. However, hexaploid wheat lacking TaZIP4-B2, only exhibits a small reduction in CO number, and still has fairly regular segregation. This is due to the four copies of ZIP4 present in hexaploid wheat: one copy on each of the homoeologous group 3 chromosomes (TaZIP4-A1, TaZIP4-B1, and TaZIP4-D1) and a fourth copy on chromosome 5B (TaZIP4-B2). TaZIP4-B2 is a transduplication of a chromosome 3B locus (International Wheat Genome Sequencing Consortium [IWGSC], 2018) and most probably appeared within the Ph1 locus upon polyploidization. In wheat lacking Ph1 (and therefore TaZIP4-B2), ZIP4 copies on the homoeologous group 3 are still present, allowing CO formation, even if a small fraction occurs between non-homologous chromosomes. In the case of an allopolyploid species such as wheat, the process of homolog recognition is further complicated compared to diploids, by the presence of homoeologous chromosomes. We can hypothesize that upon polyploidization, the newly formed hexaploid wheat already had mechanisms in place for the meiotic sorting of homologs from homoeologs during the telomere bouquet. This would have provided the new allopolyploid with some fertility until improved by the transduplication of ZIP4 from 3B into the Ph1 locus, with further modification and stabilization of the meiotic process. Over time and evolution, the efficiency and stability of meiosis could be completely established. The transduplication of ZIP4 is an example of how the meiotic program could be modified in polyploids in general, and wheat in particular, during evolution. It also illustrates the requirement to study such processes directly in these crop species, where there is a potential for manipulation in breeding programs.

C-Ph1, which is a syntenic ortholog of the RA8 gene in rice, has also been proposed to contribute to the Ph1 effect on recombination (Bhullar et al., 2014). The RA8 gene in rice encodes an anther-specific BURP-domain protein expressed specifically in the tapetum, endothecium, and connective tissue, but not in pollen grains, starting from the tetrad stage and reaching the maximum level of expression at the late vacuolated-pollen stage (Ding et al., 2009). Thus, RA8 is suggested to play an important role in microspore development and dehiscence of anther (Jeon et al., 1999). Moreover, knockouts of this gene have been reported to induce male sterility (Patents WO2000026389 A3 and US20040060084). Expression profile analysis of all different copies of the C-Ph1 gene using data generated in the present study, 849 wheat RNA-seq samples now publicly available (Ramírez-González et al., 2018), and the meiotic RNA-seq libraries deposited at https://urgi.versailles.inra.fr/files/RNASeqWheat/Meiosis/ reveals that the C-Ph1 copy on 5D (rather than on 5B) is by far the most dominantly expressed. In addition, the newly generated wheat genome assembly reveals that the VIGS hairpin construct used for C-Ph1 silencing (Bhullar et al., 2014), and which yielded sterility phenotypes, was designed from the wheat expressed sequence tag (EST) homolog BE498862 (448 bp), which is 100% identical to the 5D gene copy and not the 5B copy. As for the expression pattern, the C-Ph1 copy on 5B shows no meiotic expression (<0.5 TPM), being mostly expressed afterward during pollen formation. The C-Ph1 copy on 5D is expressed during meiosis at a very low level (<4 TPM) compared to its expression in anthers during the heading stage (>1700 TPM), exhibiting a similar expression profile to the C-Ph1 ortholog in rice RA8. These observations explain why our deletion covering the 5B copy of C-Ph1 did not exhibit meiotic phenotype or sterility (Roberts et al., 1999; Al-Kaff et al., 2008), and suggest that C-Ph1 is actually involved in microspore development and dehiscence of anther. Finally, Ph1 is the dominant gene suppressing homoeologous CO within the wheat genome. Yet neither the presence of wild type C-Ph1, nor that of any other gene could suppress homoeologous CO induced by mutating ZIP4 on 5B (TaZIP4-B2), being the same level of homoeologous CO to that observed in ph1b deletion mutants (Rey et al., 2017, 2018a). We suggest that the use of the term C-Ph1 for this gene is therefore misleading and should be replaced with a more appropriate description.