**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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# Magnesium Increases Homoeologous Crossover Frequency During Meiosis in *ZIP4* (*Ph1* Gene) Mutant Wheat-Wild Relative Hybrids

María-Dolores Rey 1 \*, Azahara C. Martín1 , Mark Smedley 1 , Sadiye Hayta1 , Wendy Harwood1 , Peter Shaw2 and Graham Moore1

#### *Edited by:*

Tomás Naranjo, Complutense University of Madrid, Spain

#### *Reviewed by:*

Andreas Houben, Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Germany Fangpu Han, Institute of Genetics and Developmental Biology (CAS), China

*\*Correspondence:*

María-Dolores Rey<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

*Received:* 08 March 2018 *Accepted:* 03 April 2018 *Published:* 20 April 2018

#### *Citation:*

Rey M-D, Martín AC, Smedley M, Hayta S, Harwood W, Shaw P and Moore G (2018) Magnesium Increases Homoeologous Crossover Frequency During Meiosis in ZIP4 (Ph1 Gene) Mutant Wheat-Wild Relative Hybrids. Front. Plant Sci. 9:509. doi: 10.3389/fpls.2018.00509 1 Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich, United Kingdom, 2 Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich, United Kingdom

Wild relatives provide an important source of useful traits in wheat breeding. Wheat and wild relative hybrids have been widely used in breeding programs to introduce such traits into wheat. However, successful introgression is limited by the low frequency of homoeologous crossover (CO) between wheat and wild relative chromosomes. Hybrids between wheat carrying a 70 Mb deletion on chromosome 5B (ph1b) and wild relatives, have been exploited to increase the level of homoeologous CO, allowing chromosome exchange between their chromosomes. In ph1b-rye hybrids, CO number increases from a mean of 1 CO to 7 COs per cell. CO number can be further increased up to a mean of 12 COs per cell in these ph1b hybrids by treating the plants with Hoagland solution. More recently, it was shown that the major meiotic crossover gene ZIP4 on chromosome 5B (TaZIP4-B2) within the 70 Mb deletion, was responsible for the restriction of homoeologous COs in wheat-wild relative hybrids, confirming the ph1b phenotype as a complete Tazip4-B2 deletion mutant (Tazip4-B2 ph1b). In this study, we have identified the particular Hoagland solution constituent responsible for the increased chiasma frequency in Tazip4-B2 ph1b mutant-rye hybrids and extended the analysis to Tazip4-B2 TILLING and CRISPR mutant-Ae variabilis hybrids. Chiasma frequency at meiotic metaphase I, in the absence of each Hoagland solution macronutrient (NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O) was analyzed. A significant decrease in homoeologous CO frequency was observed when the Mg2+ ion was absent. A significant increase of homoeologous CO frequency was observed in all analyzed hybrids, when plants were irrigated with a 1 mM Mg2+ solution. These observations suggest a role for magnesium supplementation in improving the success of genetic material introgression from wild relatives into wheat.

Keywords: wheat, wild relatives, magnesium, *Ph1* locus, *TaZIP4-B2*, homoeologous crossover, CRISPR/Cas9 system

## INTRODUCTION

Despite possessing related ancestral genomes (genome AABBDD), bread wheat behaves as a diploid during meiosis. Deletion of chromosome 5B in tetraploid and hexaploid wheat results in a level of incorrect chromosome pairing and exchange, visualized as a low level of multivalents at metaphase I, and homoeologous crossovers (COs) between related chromosomes in wheat-wild relative hybrids (Riley and Chapman, 1958; Sears and Okamoto, 1958). From these observations, it was proposed that chromosome 5B carries a locus termed Pairing homoeologous 1 (Ph1), which evolved on wheat's polyploidisation and restricted chromosome pairing and COs to true homologs (Riley and Chapman, 1958). A hexaploid wheat cv. Chinese Spring (CS) line carrying a 70 Mb deletion on the long arm of chromosome 5B (ph1b) has been exploited over the last 40 years to allow exchange between wild relative and wheat chromosomes. Recently, it was shown that on wheat's polyploidisation, a segment of 3B carrying the major crossover gene ZIP4 and a block of heterochromatin, duplicated and inserted between two CDK2-like genes within a cluster of CDK2-like and methyl-transferase genes (Griffiths et al., 2006; Al-Kaff et al., 2008; Martín et al., 2014, 2017). Using exploitation of TILLING mutants, it was shown that the duplicated ZIP4 gene (TaZIP4-B2) within this cluster, both promotes homologous CO and restricts homoeologous CO (Rey et al., 2017). Therefore, TaZIP4-B2 within the 70 Mb ph1b deletion region is responsible for the effect on homoeologous CO in wheat-wild relative hybrids, and as such the ph1b line can be described as a complete-deletion (or complete loss-of-function) mutant of Tazip4-B2 (Tazip4-B2 ph1b mutant). In terms of the effect on chromosome synapsis/pairing, cell biological studies reveal that the ph1b deletion in wheat has little effect, with most synapsis occurring during clustering of the telomeres as a bouquet. Furthermore, in wheat-wild relative hybrids, which only possess homoeologues, the ph1b deletion also has little effect on the level of synapsis, except that most pairing occurs after dispersal of the telomere bouquet. In wheat itself, a few chromosomes also undergo delayed pairing until after dispersal of the bouquet, with the subsequent incorrect pairing leading to the low level of multivalents observed at metaphase I (Martín et al., 2014, 2017).

For the last 40 years, the wheat CS ph1b deletion line has been exploited in crosses with wild relatives to allow exchange between chromosomes at meiosis. As indicated previously, in these hybrids, the extent of chromosome synapsis is similar whether the line carries the ph1b deletion or not. Moreover, on the synapsed chromosomes, similar numbers of MLH1 sites (normally a marker for CO), are observed (Martín et al., 2014). However significant site CO frequency is only observed in those hybrids carrying the ph1b deletion. However even in this case, the frequency of resulting COs still does not reflect the number of available MLH1 sites (Martín et al., 2014). This implies that there is potential for increased processing of MLH1 sites into COs. Fortuitously, it has been observed that a nutrient solution (Hoagland's solution) added to the soil when Tazip4-B2 ph1b mutant-rye hybrids are growing resulted in increased CO frequency, although it was not known which nutrient component was responsible for the effect (Martín et al., 2017).

Mineral elements are essential nutrients for plants to complete their life cycle. They are classified into macro and micronutrients, which are required in relatively large and small amounts, respectively (Hoagland and Arnon, 1950). The importance of each of these macronutrients has been reported in numerous physiological processes, such as plant growth, cell division, and metabolism (Huber, 1980; Maathuis, 2009). However, limited studies have been performed as to their effect on meiosis. Early studies have previously reported that alterations of external factors, such as temperature, or nutrient composition, can produce profound effects on chiasma frequency (Grant, 1952; Wilson, 1959; Law, 1963; Bennett and Rees, 1970; Fedak, 1973).

The main objective of the present study was to determine whether a specific macronutrient present in the Hoagland solution was responsible for the observed increased homoeologous CO frequency in Tazip4-B2 ph1b mutantrye hybrids described in Martín et al. (2017). We also analyzed whether this macronutrient increased homoeologous CO frequency in each of the Tazip4-B2 ph1b (complete deletion), TILLING (point mutation) and CRISPR (partial deletion) mutant-Ae. variabilis hybrids.

## MATERIALS AND METHODS

#### Plant Material

Plant material used in this study included: Triticum aestivum (2n = 6x = 42; AABBDD) cv. Chinese Spring Tazip4- B2-ph1b mutant line (Sears, 1977); Triticum aestivum cv. Chinese Spring-rye hybrids—crosses between the Tazip4-B2 ph1b mutant line hexaploid wheat and rye [Secale cereal cv. Petkus (2n = 2x = 14; RR)]; Triticum aestivum cv. Chinese Spring-Aegilops variabilis hybrids—crosses between Tazip4-B2 ph1b mutant and Ae. variabilis Eig. (2n = 4x = 28; UUSv S v ); Triticum aestivum cv. Cadenza-Ae. variabilis hybrids—crosses between Cad1691 and Cad0348, Tazip4-B2 TILLING mutants and Ae. variabilis (Krasileva et al., 2017; Rey et al., 2017); and Triticum aestivum cv. Fielder-Ae. variabilis hybrids—crosses between Tazip4-B2 CRISPR mutant and Ae. variabilis (see Production of TaZIP4-B2 knock-out using RNA-guided Cas9, section Materials and Methods).

#### Nutrient Solution Treatments

The total number of plants used in this work is described in **Table S1**. All seedlings were vernalized for 3 weeks at 7◦C under a photoperiod of 16 h light/8 h dark, and then transferred to a controlled environmental room until meiosis (approximately 2 months later for all genotypes used in this study). The growth conditions were 16 h/8 h, light/dark photoperiod at 20◦C day and 15◦C night, with 70% humidity. At least 2 weeks before meiosis, irrigation of plants with a Hoagland solution (100 mL per plant) was commenced following the method previously described in Martín et al. (2017). Briefly, plants were irrigated once a week with a Hoagland solution (100 mL) from the stem elongation stage of the vegetative stage (stage 7–8, Feeke's scale). The composition of the Hoagland solution was: (macronutrients) KNO3 (12 mM), Ca (NO3)2·4H2O (4 mM), NH4 H2PO4 (2 mM), Mg SO4·7H2O (1 mM); and (micronutrients) NaFe-EDTA (60 mM), KCl (50µM), H3BO3 (25µM), Mn SO4·H2O (2µM), Zn SO4 (4µM), Cu SO4·5H2O (0.5µM), H2MoO4 (0.5µM). Four treatments were carried out to analyze the effect of the absence of NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O from the Hoagland solution on homoeologous CO frequency in Tazip4-B2-ph1b mutant-rye hybrids. For each treatment, a different Hoagland solution was prepared in the absence of each macronutrient (NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O). Moreover, the effect of the presence of only Mg SO4·7H2O (Mg SO4·7H2O is designed as Mg2+ in the manuscript) in only water rather than in Hoagland solution on CO frequency was also analyzed in Tazip4-B2-ph1b mutant-rye hybrids in comparison to the effects of Hoagland solution. Also, two different concentrations of Mg2+ (1 and 2 mM of Mg2+ in water) were used to assess the homoeologous CO frequency in Tazip4-B2-ph1b mutant-rye hybrids. The treatment with either Mg2+ in water alone or Hoagland solution in Tazip4- B2 ph1b mutant-Ae. variabilis, and Tazip4-B2 TILLING and CRISPR mutant-Ae. variabilis hybrids was also assessed.

Assessment of the addition of Mg2+ in water alone on homoeologous CO frequency was also made on non-irrigated plants, by injecting into Tazip4-B2 ph1b mutant-rye hybrids tillers a solution containing 1 mM Mg2+ in water (0.5 mL per spike) just above every spike at the stage 9 on the Feekes's scale (three spikes with water alone and three spikes with Mg2+ in water). All spikes were analyzed 24–48 h after the injection.

#### Feulgen-Stained Analysis

After either irrigating with Hoagland or Mg2+ solution, or injecting the Mg2+ solution, tillers were harvested when the flag leaf was completely emerged, and only anthers at meiotic metaphase I were collected and fixed in 100% ethanol/acetic acid 3:1 (v/v). The anthers used in this study were taken from spikelets in the lower half of the spike. From each spikelet, the 2 largest florets (on opposing sides of the floret) were used. From each dissected floret, one of the three synchronized anthers was squashed in 45% acetic acid/distilled water (v/v) and the meiocytes assessed for being at meiotic metaphase I by observation under a phase contrast microscope [LEICA DM2000 microscope (LeicaMicrosystems, http://www. leica-microsystems.com/)]. The two remaining anthers were left then fixed in 100% ethanol/acetic acid 3:1 (v/v) for cytological analysis of meiocytes. The anthers were incubated in ethanol/acetic acid at 4◦C for at least 24 h. Cytological analysis of meiocytes at metaphase I was performed using Feulgen reagent as previously described in Sharma and Sharma (2014). Metaphase I meiocytes were observed under a phase contrast microscope equipped with a Leica DFC450 camera and controlled by LAS v4.4 system software (Leica Biosystems, Wetzlar, Germany). The digital images were used to determine the meiotic configurations of the meiocytes by counting the number of univalents, rod (1 chiasma) and ring (2 chiasmata) bivalents and multivalents (trivalents (1– 2 chiasmata), tetravalents (3 chiasmata) and pentavalents (4 chiasmata)). Two different methods depending on the number of chiasma (single or double chiasmata) were used to calculate chiasma frequency per meiocyte (see **Figure S1** for examples of the scored structures). Images were processed using Adobe Photoshop CS5 (Adobe Systems Incorporated, US) extended version 12.0 × 64.

#### Production of *TaZIP4-B2* CRISPR Mutants Using RNA-Guided Cas9

Three single guide RNAs (sgRNA) were designed manually to specifically target TaZIP4-B2. These guides were in the limited regions where there was sufficient variation between ZIP4 on 5BL and homoeologous group 3 chromosomes (**Figure 3**). The specific guides were: Guide 4: 5′GATGAG CGACGCATCCTGCT3′ , Guide 11: 5′GATGCGTCGCTC ATCCTCCG3′ and Guide 12: 5′GAAGAAGGATGCGGCC TTGA3′ (**Figure 3**). Two constructs were assembled using standard Golden Gate assembly (Werner et al., 2012) with each construct containing the Hygromycin resistance gene under the control of a rice Actin1 promoter, Cas9 under the control of the rice ubiquitin promoter and two of the sgRNAs each under the control of a wheat U6 promoter (**Figure 3**). Construct 1 contained guides 4 and 12 and construct 2 contained guides 11 and 12. To produce each gRNA, a PCR reaction was performed using Phusion High-Fidelity Polymerase (Thermo Scientific M0530S) with a forward primer containing the gRNA sequence, and a standard reverse primer 5′TGTGGT CTCAAGCGTAATGCCAACTTTGTAC3′ using the plasmid pICSL70001::U6p::gRNA (Addgene plasmid 46966) as template. Each gRNA was cloned individually into the level 1 vectors pICH47751 (gRNA4 & 11) and pICH47761 (gRNA12). Level 1 construct pICH47802-RActpro::Hpt::NosT (selection maker), pICH47742-ZmUbipro::Cas9::NosT and the gRNAs were then assembled in the binary Level 2 vector pGoldenGreenGate (pGGG) a Golden Gate compatible vector based on pGreen (Hellens et al., 2000) (**Figure 3**).

The two constructs were introduced to T. aestivum cv. Fielder by Agrobacterium-mediated inoculation of immature embryos. 450 immature embryos were inoculated with Agrobacterium strain AGL1 containing each construct. Briefly, after 3 days co-cultivation with Agrobacterium, immature embryos were selected on 15 mg/l hygromycin during callus induction for 2 weeks and 30 mg/l hygromycin for 3 weeks in the dark at 24◦C on Murashige and Skoog medium (MS; Murashige and Skoog, 1962) 30 g/l Maltose, 1.0 g/l Casein hydrolysate, 350 mg/l Myoinositol, 690 mg/l Proline, 1.0 mg/lThiamine HCl (Harwood et al., 2009) supplemented with 2 mg/l Picloram, 0.5 mg/l 2,4- Dichlorophenoxyacetic acid (2,4-D). Regeneration was under low light (140 µmol.m-2.s-1) conditions on MS medium with 0.5 mg/l Zeatin and 2.5 mg/l CuSO45H2O.

Primary transgenic plants (T0) were analyzed by PCR across the region of interest. The sequences for the forward and reverse primers used for the screening in T0 were 5′GCCGCCAT GACGATCTCCGAG3′ and 5′GGACGCGAGGGACGCGAG3′ , respectively (Rey et al., 2017), followed by direct sequencing. The PCR was performed using RedTaq ReadyMix PCR Reaction Mix (Sigma, St. Louis, MO, USA; R2523) according to the manufacturer's instructions. PCR conditions were: 3 min 95C, 35 cycles of 15 s at 95C, 15 s at 58C and 30 s at 72C. T0 plants with edits in TaZIP4-B2 were progressed to the T1 generation and 24 T1 seedlings from each original T0 plant were analyzed in the same way for the presence of edits.

#### Statistical Analyses

Statistical analyses were performed using STATISTIX 10.0 software (Analytical Software, Tallahassee, FL, USA). Analysis of variance (ANOVA) in Tazip4-B2 ph1b mutant-rye hybrids, Tazip4-B2 TILLING mutant-Ae. variabilis hybrids and Tazip4- B2 CRISPR mutant-Ae. variabilis hybrids was based on a completely randomized design. Several transformations were carried out: tangent (ring bivalents), arcsine (trivalents), and logarithm (double CO) transformations in the analysis of the effect of absence of each macronutrients in homoeologous CO frequency in Tazip4-B2 ph1b mutant-rye hybrids; exponential (ring bivalents) transformation in Tazip4-B2 ph1b mutant-Ae. variabilis hybrids; exponential (rod bivalents, rings bivalents and trivalents) transformation in Tazip4-B2 TILLING mutant (Cad1691)-Ae. variabilis hybrids; and square root (ring bivalents) and exponential (trivalents) transformations in Tazip4-B2 TILLING mutant (Cad0348)-Ae. variabilis hybrids. Means were separated using the Least Significant Difference (LSD) test with a probability level of 0.05. Both Tazip4-B2 CRISPR mutant lines and Tazip4-B2 CRISPR mutant-Ae. variabilis hybrids were analyzed by the Kruskal–Wallis test (non-parametric one-way analysis of variance). Means were separated using the Dunn's test with a probability level of 0.05.

#### RESULTS

#### Magnesium Increases Homoeologous COs in *Tazip4-B2 ph1b* Mutant-Rye Hybrids

The Tazip4-B2 ph1b mutant-rye hybrids were obtained by crosses between the hexaploid wheat cv. Chinese Spring Tazip4-B2 ph1b mutant and rye. These hybrids were used to analyse which macronutrient (NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O) present within in the Hoagland solution detailed in Martín et al. (2017) could be responsible for the increased CO number observed in the Tazip4-B2 ph1b mutant-rye hybrids. To assess the effect of the absence of each macronutrient in homoeologous CO frequency in meiotic metaphase I, we irrigated several Tazip4-B2 ph1b mutant-rye hybrids with: (1) Hoagland solution; (2) water alone; (3) Hoagland solution minus KNO3; (4) Hoagland solution minus Ca (NO3)2·4H2O; (5) Hoagland solution minus NH4 H2PO4; (6) Hoagland solution minus MgSO4·7H2O (MgSO4·7H2O is designed as Mg2+ in the manuscript) (**Table 1**). The absence of each Hoagland solution macronutrient caused a slight increase in homoeologous CO frequency, except for the treatment lacking Mg2+, where a significant decrease in homoeologous CO frequency per meiocyte was observed at meiotic metaphase I in these hybrids (**Table 1**). No significant differences in CO frequency at metaphase I were observed between hybrids treated with water alone and those

treated with the Hoagland solution minus Mg2+ [a mean of 7.91 chiasmata for hybrids treated with water alone and 8.09 chiasmata for hybrids treated with Hoagland solution minus Mg2+ (**Table 1**)].

Additionally, we scored all meiocytes for the occurrence of double chiasmata in the metaphase I chromosomal configurations (examples highlighted by arrows in **Figure S1**). When double chiasmata were considered in the chiasma frequency, a mean of 8.15 chiasmata and 8.62 chiasmata was observed respectively in Tazip4-B2 ph1b mutant-rye hybrids treated with water alone, and those treated with the Hoagland solution minus Mg2+ (**Table 1**). As expected, no significant differences were observed between the two treatments when double chiasmata were considered in these Tazip4-B2 ph1b hybrids.

Once the absence of Mg2+ was demonstrated to decrease homoeologous CO frequency in Tazip4-B2 ph1b mutant-rye hybrids, the effect of irrigating with only Mg2+ present at a final concentration of 1 mM in water rather than in the Hoagland solution, was also analyzed on homoeologous COs in Tazip4-B2 ph1b mutant-rye hybrids (**Figure 1**). Treatment with a solution containing only Mg2+ also increased homoeologous COs at metaphase I per meiocyte in these hybrids, showing no significant difference in comparison to the Hoagland solution treatment. A mean of 11.09 chiasmata was observed after treatment with 1 mM Mg2+ in water, and 10.74 chiasmata after treatment with the Hoagland solution (**Figure 1**), when a single chiasma was considered. A similar situation was seen when double chiasmata were considered: no significant differences were observed in homoeologous COs per meiocyte in Tazip4-B2 ph1b mutant-rye hybrids after treatment with either 1 mM Mg2+ or Hoagland solutions (**Figure 1**).

The concentration of Mg2+ was subsequently increased to a final concentration of 2 mM to assess whether the number of homoeologous COs could be increased further (**Figure 1**). Surprisingly, numbers of COs were reduced under these conditions (mean 11.09 for 1 mM Mg2+ and 8.84 for 2 mM Mg2+ treatments respectively, when single chiasma were considered, and 12.11 and 9.90, respectively, when double chiasmata were considered).

In addition to irrigating the plants with either Hoagland or Mg2+ solutions, we analyzed the effect of treatment with 1 mM Mg2+ in water following injection into the tillers of Tazip4-B2 ph1b mutant-rye hybrids. Injections were made just above each spike. Once again, homoeologous CO frequency was significantly increased in hybrids treated with 1 mM Mg2+ when the solution was injected into the tiller (**Table 2**). A mean of 8.98 chiasmata in hybrids treated with water alone and 10.60 chiasmata in hybrids treated with 1 mM Mg2+ was observed in the hybrids considering a single chiasma (**Table 2**) and a mean of 9.67 chiasmata and 11.30 chiasmata considering double chiasmata (**Table 2**).

#### Magnesium Increases Homoeologous COs in *Tazip4-B2 ph1b Mutant-Ae. variabilis* Hybrids

The addition of Mg2+ is thus identified as responsible for the increase in homoeologous CO at meiotic metaphase I in Tazip4- B2 ph1b mutant-rye hybrids. We then assessed the effect of 1 mM Mg2+ on T. aestivum cv. Chinese spring Tazip4-B2 ph1b mutant-Ae. variabilis hybrids. Firstly, we scored the number of univalents, bivalents and multivalents, and total chiasma frequency in this hybrid, to compare the level of chiasma

FIGURE 1 | Effect of either 1 mM or 2 mM Mg2+ on homoeologous CO frequency of T. aestivum cv. Chinese Spring Tazip4-B2 ph1b mutant-rye hybrids. (A) Frequencies of univalents, bivalents, trivalents, and chiasma frequency (single and double chiasmata) were scored at meiotic metaphase I in Tazip4-B2 ph1b mutant-rye hybrids treated with either Hoagland solution, 1 mM Mg2+ or 2 mM Mg2+solution. Values in parenthesis indicate range of variation between cells. P < 0.05 indicates significant differences according to LSD test. Different letters indicate significant differences between treatments. (B) Representative meiotic configurations of Tazip4-B2 ph1b mutant-rye hybrids. From left to right: treatment with Hoagland solution, 1 mM Mg2+or 2 mM Mg2+ solution. Bar: 20µm.

frequency to that previously reported by Kousaka and Endo (2012) in T. aestivum cv. Chinese spring-Ae. variabilis hybrids in the absence of chromosome 5B. We observed a similar chiasma frequency in our hybrid (mean 14.15 chiasmata per meiocyte), to that previously reported in T. aestivum cv. Chinese spring-Ae. variabilis hybrids in the absence of chromosome 5B (mean of 14.09 chiasmata per meiocyte), confirming a similar level of meiotic metaphase I configuration in these hybrids.

We then analyzed the effect of treatment with water alone and with either 1 mM Mg 2 + solution or complete Hoagland solution on the Tazip4-B2 ph1b mutant-Ae. variabilis hybrids. The total number of COs was significantly higher after treatment with 1 mM Mg 2 + than after treatment with water alone (without Mg 2 + control), both in the case of single chiasma and double chiasmata, showing a mean of 15.31 and 14.15 chiasmata in the case of single chiasma, and a mean of 16.54 and 15.10 chiasmata in the case of double chiasmata, respectively (**Figure 2**). The number of univalents was significantly decreased and the number of trivalents was significantly increased when the plants were treated with 1 mM Mg 2 + solution in comparison to when Mg 2 + was absent (a mean of 11.14 and 1.99, respectively, after treatment with 1 mM Mg 2 + and a mean of 12.49 and 1.60, respectively, after treatment with water alone were observed in **Figure 2**). With regard to the Hoagland solution treatment, significant differences were observed between hybrids treated with water alone and hybrids treated with Hoagland solution (**Figure 2**). Hoagland solution treatment showed the highest chiasma frequency, followed by 1 mM Mg 2 + and water alone [means of 16.53, 15.31, and 14.15 chiasmata were observed, respectively, when a single chiasma was considered, and means of 18.05, 16.54, and 15.10 chiasmata were observed, respectively, when double chiasmata were considered (**Figure 2**)].

#### Magnesium Increases Homoeologous COs in Wheat *Tazip4-B2* TILLING Mutant-*Ae. variabilis* Mutant Hybrids

Recently we reported that Tazip4-B2 TILLING mutants crossed with Ae. variabilis exhibited homoeologous COs at meiotic metaphase I (Rey et al., 2017). We therefore decided to analyse whether the level of homoeologous COs induced by Tazip4- B2 TILLING mutants was also affected by treatment with 1 mM Mg 2 + solution. To assess the effect of 1 mM Mg 2 + on homoeologous CO frequency at metaphase I, we added 100 mL per plant of a solution of either 1 mM Mg 2 + in water or Hoagland solution once a week to the soil in which these hybrids were growing. In this experiment, we analyzed both Tazip4-B2 TILLING mutant lines (Cad1691 and Cad0348) (Rey et al., 2017), crossed with Ae. variabilis. Both TILLING mutant hybrids showed a significant increase in chiasma frequency after treatment with 1 mM Mg 2 +, compared to chiasma frequency obtained in both the hybrids treated with water alone. The Tazip4-B2 TILLING mutant (Cad1691)-Ae. variabilis and the Tazip4-B2 TILLING mutant (Cad0348)-Ae. variabilis hybrids showed means of 13.41 and 13.66 single chiasma frequency,

TABLE

2

of

injecting

1

mM

Mg2+

solution

into

the

tillers

of

Tazip4-B2

ph1b

mutant-rye

hybrids.

Values in parenthesis

 indicate range of variation between cells. P < 0.05 indicates significant differences according to LSD test. Different letters indicate significant differences between treatments.

20µm.

respectively, after treatment with 1 mM Mg2+ and means of 12.21 and 12.23 single chiasma frequency, respectively, in water alone (**Table 3**; **Figure S2**). Significant differences were also observed when double chiasmata were scored in both mutant lines (**Table 3**; **Figure S2**). Numbers of univalents and trivalents were also affected by treatment with 1 mM Mg2+ in both mutant lines as in the Tazip4-B2 ph1b mutant-Ae. variabilis hybrids described in the previous section. Numbers of univalents were significantly decreased both in Tazip4-B2 TILLING mutant (Cad1691)- Ae. variabilis and Tazip4-B2 TILLING mutant (Cad0348)- Ae. variabilis hybrids treated with 1 mM Mg2+ [means of 12.74 and 12.11 univalents respectively with Mg2+ and means of 14.74 and 14.63 univalents respectively with water alone (**Table 3**)]. Numbers of trivalents were significantly increased both in wheat (Cad1691)-Ae. variabilis and wheat (Cad0348)- Ae. variabilis hybrids, after treatment with 1 mM Mg2+ [means of 1.63 and 1.93 trivalents respectively with Mg2+, and means of 1.05 and 1.27 trivalents respectively with water alone (**Table 3**)].

Finally, we assessed the effect of treating with Hoagland solution and with water alone, finding significant differences in homoeologous COs between the two treatments, both in Tazip4- B2 TILLING mutant (Cad1691)-Ae. variabilis and Tazip4-B2 TILLING mutant (Cad0348)-Ae. variabilis hybrids. Numbers of univalents and trivalents were also affected to the same extent (**Table 3**). In the Tazip4-B2 TILLING mutant (Cad1691)- Ae. variabilis hybrid, means of 14.74 univalents and 1.05 trivalents were observed in hybrids treated with water alone, and means of 11.94 univalents and 1.50 trivalents observed in hybrids treated with Hoagland solution (**Table 3**). In the Tazip4-B2 TILLING mutant (Cad0348)-Ae. variabilis hybrid, means of 14.63 univalents and 1.27 trivalents were observed in hybrids with water alone and means of 12.48 univalents and 1.76 trivalents in hybrids treated with Hoagland solution (**Table 3**).

### Phenotypic Analysis of *Tazip4-B2* Mutants Generated by CRISPR/Cas9 System

Firstly, eighty-one primary transgenic plants (T0) were analyzed by PCR followed by direct sequencing. Four plants were identified with edits in the target region. One plant had a perfect 115 bp deletion between guides G11 and G12. Twentyfour T1 plants from this line were screened and 5 homozygous edited plants with the 115 bp deletion were recovered. These plants were used to score the number of univalents, bivalents, and multivalents, and total chiasma frequency in the Tazip4-B2 mutant CRISPR lines (**Figure 3**). Wild-type Fielder lines were used as control plants (**Figure 3**). The Tazip4-B2 CRISPR mutant lines exhibited a significant reduction in ring bivalents, from a mean of 18.33 to 14.84 in the wild-type Fielder and CRISPR mutant lines respectively (**Figure 3**). A significant increase in the number of univalents and rod bivalents was also observed, from means of 0.51 univalents and 2.38 rod bivalents in the wild-type Fielder line, to means of 1.16 univalents and 4.93 rod bivalents in the CRISPR mutant lines (**Figure 3**). This indicates a significant reduction in homologous COs in these Tazip4-B2 mutant lines (**Figure 3**). Chiasma frequency decreased from a mean of 39.07 single chiasma and 40.50 double chiasmata in the wild-type Fielder line, to a mean of 35.55 single chiasma and 37.11 double chiasmata in the Tazip4-B2 CRISPR mutant (**Figure 3**).

#### Magnesium Also Increases Homoeologous COs in Wheat *Tazip4-B2* CRISPR Mutant-*Ae. variabilis* Mutant Hybrids

For this study, a wild-type Fielder and a Tazip4-B2 CRISPR Fielder mutant line were crossed with Ae. variabilis to assess the level of homoeologous COs in the resulting hybrids (**Table S2**). Frequency of univalents, bivalents and multivalents, and total chiasma frequency were scored at meiotic metaphase I (**Table S2**). Tazip4-B2 CRISPR mutant hybrids exhibited a significant increase in single chiasma frequency, from a mean of 3.15 in the wild-type Fielder-Ae. variabilis hybrid to 16.66 in the Tazip4-B2 CRISPR-Ae. variabilis hybrid (**Table S2**). Double chiasma frequency was also increased in the Tazip4-B2 CRISPR mutant hybrids (**Table S2**). There was also a similar increase in the chiasma frequency to that reported previously in Tazip4-B2 TILLING-Ae. variabilis hybrids (Rey et al., 2017).

Having observed the effect of treatment with Mg2+ on homoeologous CO frequency in the Tazip4-B2 TILLING mutant hybrids, we also analyzed the effect of this ion on Tazip4-B2 CRISPR mutants-Ae. variabilis hybrids. We added 100 mL of a solution of 1 mM Mg2+ in water or Hoagland solution once a week to the soil in which the hybrids were growing. As expected, the addition of nutrients to these mutant hybrids caused a significant increase in chiasma frequency (**Table 4**; **Figure S2**). Tazip4-B2 CRISPR-Ae. variabilis hybrids treated with water alone exhibited means of 16.66 single chiasma frequency and 18.10 double chiasma frequency. Addition of 1 mM Mg2+ caused a significant increase in chiasma frequency of these mutant hybrids (means of 17.67 and 18.75 single and double chiasma frequency respectively) (**Table 4**). Also, the addition of Hoagland solution increased the homoeologous COs in these Tazip4-B2 CRISPR hybrids. Means of 18.34 and 19.82 single and double chiasma frequency respectively, were observed in those plants treated with Hoagland solution (**Table 4**).

#### DISCUSSION

Introgression of genetic material from relative species into bread wheat has been used in plant breeding for over 50 years, although classical plant breeding methods to introgress wild relative segments into wheat are both inefficient and time consuming (Ko et al., 2002). Recent availability of SNP based arrays, combined with classical cytogenetic approaches, significantly enhanced our ability to exploit wild relatives (King et al., 2017a,b), using lines carrying a deletion of either the whole of chromosome 5B, or a smaller 70 Mb segment (ph1b) (Riley and Chapman, 1958; Sears and Okamoto, 1958; Sears, 1977), to increase the level of homoeologous crossovers between wild relatives and wheat chromosomes. Recombination between wild relative chromosomes and wheat chromosomes is, however, still limited. Thus, there is a need to find abiotic or biotic treatments such as temperature, nutritional availability, DNAdamaging agents, among others (Lambing et al., 2017) to enhance recombination. Martín et al. (2017) recently reported an alternative tool to increase CO number in Tazip4-B2 ph1b mutant-rye hybrids, using the addition of a Hoagland solution

2+

1 mM Mg

 or Hoagland solution. Values in parenthesis

significant differences between treatments.

 indicate range of variation between cells. P < 0.05 indicates significant differences according to LSD test. \*This data published in Rey et al. (2017). Different letters indicate

indicate range of variation between cells. P < 0.05 indicates significant differences according to Dunn's test. Different letters indicate significant differences between treatments. (F) Representative meiotic metaphase I configurations of wild-type Fielder and Tazip4-B2 CRISPR Fielder mutants. Left: wheat cv. Fielder and right: Tazip4-B2 CRISPR mutant. Bar: 20µm.

to the soil in which the plants are grown. Martín et al. (2017) also showed that the presence of the Hoagland solution did not affect the homoeologous CO number in wild-type wheat-rye hybrids.

Here, we report the successful identification of the particular Hoagland solution constituent responsible for the observed increase in homoeologous CO frequency. After analyzing Tazip4- B2 ph1b mutant-rye hybrids in the absence of each separate Hoagland solution macronutrient, we observed a significant reduction in homoeologous CO frequency when the Mg2+ ion was absent. This suggests that the Mg2+ ion is mainly responsible for the effect of Hoagland solution on homoeologous COs described previously by Martín et al. (2017). These observations were obtained after cytogenetic analysis of meiotic configurations at meiotic metaphase I. The analysis involved scoring single and double chiasmata in the chromosomal structures (**Figure S1**). Single chiasma counting has commonly been used in many

 Hoagland solution. Values in parenthesis indicate range of variation between cells. P < 0.05 indicates significant differences according to LSD test. Different letters indicate significant differences between treatments.

> 1 mM Mg

2+ or studies to measure chiasma frequency in wheat (Dhaliwal et al., 1977; Sears, 1977; Roberts et al., 1999). However, other studies

have suggested that double chiasmata may occur in these chromosomal configurations (Gennaro et al., 2012; Dreissig et al., 2017). Double chiasmata were considered in the present study, as a high number of MLH1 sites were previously reported in Tazip4-B2 ph1b mutant-rye hybrids in Martín et al. (2014). In our studies, up to 19 chiasmata were scored in Tazip4-B2 ph1b mutant-rye hybrids, which is similar to the number of MLH1 sites observed previously (Martín et al., 2014).

The effect of treatment with a solution of 1mM Mg2+ in water, was analyzed to confirm whether that the Mg2+ ion was responsible for the increase in homoeologous COs observed in these hybrids. The effect of treatment with this solution was assessed either by irrigation of, or injection into Tazip4-B2 ph1b mutant-rye hybrids. Surprisingly, both methods of application increased homoeologous CO frequency in the Tazip4-B2 ph1b mutant-rye hybrids. Thus, the results from the injection method of application suggested that the 1 mM Mg2+ concentration was directly responsible for the increased homoeologous CO effect seen in the Tazip4-B2 ph1b mutant-rye hybrids, rather than through indirect effects on the plant growth or development. However, homoeologous CO frequency was decreased when the Mg2+ concentration was increased further (**Figure 1**). This reduction in COs was associated with a significant increase in the number of univalents, and decrease in the number of ring bivalents and trivalents.

A recent study revealed that TaZIP4-B2 within the 5B region defined by the 70 Mb ph1b deletion, was responsible for the suppression of homoeologous COs in hybrids (Rey et al., 2017). Tazip4-B2 TILLING mutants (one with a missense mutation and another with a nonsense mutation), when crossed with Ae. variabilis, exhibit similar levels of homoeologous CO that observed in ph1b-Ae. variabilis hybrids (Rey et al., 2017). It was therefore important to assess the effect of 1 mM Mg2+ solution on these Tazip4-B2 TILLING mutant-Ae. variabilis hybrids to confirm that the effect was associated with Tazip4-B2, and that the Mg2+ effect could also be observed in a different hybrid. Moreover, we also applied the CRISPR/Cas9 genome editing system in hexaploid wheat cv. Fielder to the mutant TaZIP4-B2 to compare its mutant phenotype with those observed in TILLING mutant lines, and their Ae. variabilis hybrids. Tazip4-B2 CRISPR mutants showed a significant decrease in homologous COs compared to control plants (TaZIP4-B2 wild type wheat), similar to that already reported for Tazip4-B2 TILLING mutants (Rey et al., 2017). Also, as expected, a significant increase was observed in Tazip4-B2 CRISPR mutant-Ae. variabilis hybrids, similar to that observed in both ph1b-Ae. variabilis and Tazip4-B2 TILLING mutant-Ae. variabilis hybrids. Furthermore, the addition of 1 mM Mg2+ to all these hybrids increased the frequency of homoeologous CO. This confirms that the Mg2+ effect is associated with Tazip4-B2, and occurs in different hybrids. The only difference observed with the Tazip4-B2 CRISPR and TILLING mutants was the occurrence of multivalents in the CRISPR mutants compared to the TILLING mutants (**Figure 3** and Rey et al., 2017). This suggests that

TaZIP4-B2 not only promotes homologous COs and restricts homoeologous COs, but also contributes to the efficiency of homologous pairing. We hypothesize that the CRISPR deletion disrupts more of the TaZIP4-B2 function than the TILLING mutants. Interestingly, in rice, ZIP4 mutants have previously been reported to show a delay in completing homologous synapsis (Shen et al., 2012), however, in that diploid species, this does not lead to homoeologous COs because only homologs are present. However, in the ph1b mutant, delayed pairing of some homologs is observed until after the telomere bouquet, allowing some subsequent homoeologous pairing to take place. This delayed pairing of homologs in the ph1b mutant is consistent with a ZIP4 mutant phenotype.

Magnesium is one of the most important nutrients, mainly involved in the general promotion of plant growth and development. In terms of CO function, Mg2+ may affect multiple proteins in the class I interference crossover pathway either in a positive or negative manner. For example, recent studies have suggested that Mg2+is required for the endonuclease activity of the MLH1-MLH3 heterodimer (Rogacheva et al., 2014). The MLH1-MLH3 heterodimer shows a strong preference for HJs in the absence of Mg2+ (Ranjha et al., 2014). Whatever the target, our present study reveals that homoeologous COs can be increased by the 1 mM Mg2+ treatment of Tazip4-B2 (ph1b, TILLING or CRISPR derived) mutant-wild relative hybrids. Thus, this treatment can be used as a tool to enhance the introgression of wild relative traits into wheat.

#### AUTHOR CONTRIBUTIONS

M-DR, AM, PS, and GM: conceived and designed the study; MS, SH, and WH: participated in the development of the Tazip4- B2 mutant in bread wheat cv. Fielder by CRISPR/Cas9 system; M-DR: analyzed the research results and wrote the first draft; PS

#### REFERENCES

and GM: modified the paper. All authors have read and approved the final version of the manuscript.

## ACKNOWLEDGMENTS

The authors thank Ali Pendle (John Innes Centre, UK) for her valuable comments in the writing of the manuscript. This work was supported by the UK Biotechnology and Biological Research Council (BBSRC), through a grant part of the Designing Future Wheat (DFW) Institute Strategic Programme (BB/P016855/1), three grants (Grant BB/J004588/1; Grant BB/M009599/1; Grant BB/J007188/1); and by a Marie Curie Fellowship Grant (H2020- MSCA-IF-2015-703117).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 00509/full#supplementary-material

Figure S1 | Chromosomal configurations with single chiasma or double chiasmata highlighted with arrows. These structures marked by an arrow were counted as either single or double chiasmata in all analyzed meiocytes. Both datasets are shown in all analyzed genotypes. Bar: 20µm.

Figure S2 | Representative meiotic configurations of Triticum aestivum cv. Cadenza (Cad1691-Tazip4-B2 mutant)-Ae. variabilis (A) and Triticum aestivum cv. Cadenza (Cad0348-Tazip4-B2 mutant)-Ae. variabilis (B) and wheat Tazip4-B2 CRISPR mutant-Ae. variabilis mutant (C) hybrids. From left to right: water alone, treated with either 1 mM Mg2+ or Hoagland solution. Bar: 20µm.

Table S1 | Genotypes and number of plants used for analyzing the effect of a nutrient solution in homoeologous CO frequency in wheat and its relative species.

Table S2 | Frequencies of univalents, bivalents, multivalents and chiasma frequency (single and double chiasmata) were scored at meiotic metaphase I in wheat Tazip4-B2 CRISPR mutant-Ae. variabilis hybrids. Values in parenthesis indicate range of variation between cells. P < 0.05 indicates significant differences according to Dunn's test. Different letters indicate significant differences between treatments.

and breeding prospects. Mol. Breed. 30, 149–167. doi: 10.1007/s11032-011- 9606-6

aestivum and the effect of the gametocidal genes. Ann. Bot. 121, 229–240. doi: 10.1093/aob/mcx149

identified two lines exhibiting homoeologous crossover in wheat-wild relative hybrids. Mol. Breed. 37:95. doi: 10.1007/s11032-017-0700-2

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Rey, Martín, Smedley, Hayta, Harwood, Shaw and Moore. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Variable Patterning of Chromatin Remodeling, Telomere Positioning, Synapsis, and Chiasma Formation of Individual Rye Chromosomes in Meiosis of Wheat-Rye Additions

#### Tomás Naranjo\*

Departamento de Genética, Fisiología y Microbiología, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain

#### Edited by:

Simon Gilroy, University of Wisconsin-Madison, United States

#### Reviewed by:

Adam Lukaszewski, University of California, Riverside, United States Fangpu Han, Institute of Genetics and Developmental Biology (CAS), China

> \*Correspondence: Tomás Naranjo<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

Received: 15 March 2018 Accepted: 06 June 2018 Published: 02 July 2018

#### Citation:

Naranjo T (2018) Variable Patterning of Chromatin Remodeling, Telomere Positioning, Synapsis, and Chiasma Formation of Individual Rye Chromosomes in Meiosis of Wheat-Rye Additions. Front. Plant Sci. 9:880. doi: 10.3389/fpls.2018.00880 Meiosis, the type of cell division that halves the chromosome number, shows a considerable degree of diversity among species. Unraveling molecular mechanisms of the meiotic machinery has been mainly based on meiotic mutants, where the effects of a change were assessed on chromosomes of the particular species. An alternative approach is to study the meiotic behavior of the chromosomes introgressed into different genetic backgrounds. As an allohexaploid, common wheat tolerates introgression of chromosomes from related species, such as rye. The behavior of individual pairs of rye homologues added to wheat has been monitored in meiotic prophase I and metaphase I. Chromosome 4R increased its length in early prophase I much more than other chromosomes studied, implying chromosome specific patterns of chromatin organization. Chromosome conformation affected clustering of telomeres but not their dispersion. Telomeres of the short arm of submetacentric chromosomes 4R, 5R, and 6R failed more often to be included in the telomere cluster either than the telomeres of the long arms or telomeres of metacentrics such as 2R, 3R, and 7R. The disturbed migration of the telomeres of 5RS and 6RS was associated with failure of synapsis and chiasma formation. However, despite the failed convergence of its telomere, the 4RS arm developed normal synapsis, perhaps because the strong increase of its length in early prophase I facilitated homologous encounters in intercalary regions. Surprisingly, chiasma frequencies in both arms of 4R were reduced. Similarly, the short arm of metacentric chromosome 2R often failed to form chiasmata despite normal synapsis. Chromosomes 1R, 3R, and 7R showed a regular meiotic behavior. These observations are discussed in the context of the behavior that these chromosomes show in rye itself.

Keywords: chromatin remodeling, telomere dynamics, synapsis, chiasmata, rye, wheat, FISH

## INTRODUCTION

Bivalents formed during meiotic prophase I are essential to halve the chromosome number. Bivalent formation requires that homologous chromosomes, mainly located in separate nuclear territories in premeiotic interphase nuclei (Bass et al., 2000; Maestra et al., 2002), move to find one another. In many species, concomitant with a chromatin remodeling process that causes a

considerable chromosome elongation during leptotene, telomeres undergo an oriented migration and converge in a tight cluster in a small area of the nuclear envelop. This suprachromosomal meiotic configuration, the so-called bouquet, facilitates chromosome interactions that culminate in the identification of the homologous partner (Bass et al., 1997, 2000; Niwa et al., 2000; Trelles-Sticken et al., 2000; Cowan et al., 2001; Scherthan, 2001). The bouquet is disorganized once homologues undergo synapsis.

A programed production of double-strand DNA breaks (DSBs) catalyzed by Spo11 in conjunction with other proteins triggers the initiation of chromosome interactions (Keeney et al., 1997; Neale and Keeney, 2006). DSBs are resected to generate 3 0 single-strand DNA overhangs that bind recombinases RAD51 and DMC1. These nucleoprotein filaments invade a doublestrand DNA stretch of the homologous chromosome to find its complementary strand (Hunter and Kleckner, 2001). During leptotene, chromosomes form the axial element, a proteinrich backbone that will keep the sister chromatids together until the second division. The search of a repair template in the homologous chromosome is instigated by proteins of the axis such as Hop1-Red1 in budding yeast, ASY1-ASY3 in Arabidopsis and PAIR2-PAIR3 in rice (Hollingsworth and Byers, 1989; Thompson and Roeder, 1989; Caryl et al., 2000; Nonomura et al., 2006; Wang et al., 2011; Ferdous et al., 2012). The identification of the homologous partner in the invaded chromatid is necessary for chromosome pairing and synapsis in many organisms (Roeder, 1997; Baudat et al., 2000; Romanienko and Camerini-Otero, 2000). Then, homologues become aligned, form the tripartite synaptonemal complex (SC) during zygotene, and are fully synapsed at pachytene (Page and Hawley, 2004). The SC maintains homologues in close juxtaposition along their length and serves as a scaffold for factors of the recombinational repairing machinery (Zickler and Kleckner, 2015). The genetic control of the repairing machinery ensures a minimum of one crossover (CO) per homologous pair. However, repair of the majority (95%) of DSBs produced in plants and animals culminates in a non-crossover (NCO) (Higgins et al., 2014). The SC disassembles at diplotene, once COs formation is completed, and chromatin undergoes a progressive condensation. Meanwhile, homologues remain physically connected by chiasmata, the cytological expression of COs, until their disjunction in anaphase I.

Deviations from this meiotic prophase I program have been reported in several organisms (reviewed in Zickler and Kleckner, 2016). In fission yeast, homologous pairing is recombinationindependent and COs are formed in the absence of SC (Ding et al., 2004). In Drosophila melanogaster females and in Caenorhabditis elegans, pairing and synapsis can occur independently of recombination (Lake and Hawley, 2012; Rog and Dernburg, 2013). However, in both cases, DSBs are produced after SC formation. The D. melanogaster males and the silk worm (Bombyx mori) females both with achiasmate meiosis, show well-aligned bivalents at metaphase I. The presence of the SC until anaphase I provides the physical connection between homologues in B. mori females (Rasmussen, 1977). B. mori males form chiasmate bivalents. In D. melanogaster males, which do not form SC, pairing initiates at a specific site in sex chromosomes, and probably in chromosome 4, but no pairing center has been found in the other two autosomes (Tsai and McKee, 2011). Sex differences in the meiotic process have been observed in other organisms such as, planarian worms (Pastor and Callan, 1952; Oakley and Jones, 1982; Oakley, 1982), Lilium and Fritillaria (Fogwill, 1958), grasshoppers (Perry and Jones, 1974), Arabidopsis thaliana (Drouaud et al., 2007), mice (Petkov et al., 2007), or humans (Hou et al., 2013).

Studies aimed at unraveling the molecular mechanisms underlying the chromosome dynamics in meiotic prophase I are based on the use of meiotic mutants and often focus on the full chromosome complement, neglecting the behavior of individual chromosomes. However, individual chromosomes may respond in different way to the general program of the meiotic cell. An apparent example of this differential behavior is the case of the sex chromosomes in the heterogametic sex of species with XX/XY, XX/X0, or ZZ/ZW. Non-homologous regions of sex chromosomes appear usually unmatched and with apparent changes in the chromatin organization during prophase I, which, in the case of mammals, is accompanied by silencing of unsynapsed chromatin (Page et al., 2006; Turner, 2013). It is also remarkable the variable chromosome behavior in spermatocytes of the planarian worm Mesostoma ehrenbergii ehrenbergii. Chiasma formation is extremely restricted: three homologous pairs form one distal chiasmata and appear as bivalents at metaphase I, while the other two pairs do not form chiasmata and appear as univalents (Croft and Jones, 1989). Chromosomes of common wheat Triticum aestivum show differences in the chiasma distribution and in their ability to form chiasmata after deletion of terminal regions (Naranjo, 2015).

An alternative approach to the use of meiotic mutants in a given species is the study of the meiotic behavior of its chromosomes when they are introgressed in the genetic background of a different species. Common wheat (genome formula, AABBDD, 2n = 6x = 42) as an allohexaploid, tolerates the addition of chromosomes from related species such as rye, Secale cereale (RR, 2n = 14). A handful of complete sets of wheatrye additions are available (Lukaszewski, 2015). These lines can be used in the study of the behavior of individual rye chromosomes both in somatic and meiotic cells, in the assignation of genetic markers to chromosomes, or in the study of genetic interactions between wheat and rye chromosomes. In addition they represent an excellent start point for the introgression of useful genes of rye into wheat.

Painting of the rye chromosomes present in each disomic addition allows to monitor their pairing and synapsis and to estimate the number of chiasmata formed (Maestra et al., 2002; Corredor and Naranjo, 2007; Naranjo and Corredor, 2008; Naranjo et al., 2010; Valenzuela et al., 2012; Naranjo, 2014, 2015). Rye chromosomes bear apparent subtelomeric heterochromatin blocks (Darvey and Gustafson, 1975), which can be visualized by fluorescence in situ hybridization (FISH) making possible to identify the position of the adjacent telomere in the meiotic bouquet (Naranjo et al., 2010; Naranjo, 2014). The position of the telomere of the short arm of chromosome 5R (5RS) at the bouquet depends of the length of the other arm of

this submetacentric chromosome. The 5RS arm of the standard chromosome 5R is much shorter than the long arm (5RL), but in a truncated chromosome 5R (del5R) lacking the last 70% distal of 5RL (del5RL) both arms have a similar length. Although the 5RS arm is the same in chromosomes 5R and del5R, its telomere fails more often in its incorporation to the telomere cluster in the standard chromosome than in the truncated one. Disturbed telomere migration has a negative effect on the development of synapsis and chiasma formation (Naranjo et al., 2010). Chromosomes 1R and 6R carry subtelomeric heterochromatin chromomeres in both ends. This allowed to verify that the end of their long arms, 1RL and 6RL, were included in the telomere cluster in almost all cells (>99%) while one or both telomeres of 1RS, or 6RS, were separated of the telomere cluster in 27 and 40% of meiocytes, respectively. Thus, incomplete telomere migration occurs more often in the short arm of the submetacentric chromosome 6R than in the short arm of the almost metacentric chromosome 1R. Failure of synapsis and chiasma formation is also relatively frequent in the 6RS arm (Naranjo, 2014). This result is consistent with the behavior of 5RS in chromosomes 5R and del5R.

The meiotic behavior of chromosomes 1R, 5R, and 6R in a wheat background suggests that the genetic and structural chromosome identity is manifested in the appearance of chromosome specific features in the development of the meiotic program. Verification of this idea implies the analysis of all individual chromosomes. In the case of rye, it remains to be studied the meiotic behavior of metacentric chromosomes 2R, 3R, and 7R and submetacentric chromosome, 4R. On the other hand, from the effect of chromosome structure on telomere clustering the question arises whether telomere dynamics during bouquet dissolution is also chromosome conformation-dependent or not. The aim of this paper is to carry out a comparative study of the dynamics of individual rye chromosomes in a wheat background during prophase I. Three different aspects of the meiotic process are concerned: (i) chromatin remodeling (ii) the telomere movement leading to the formation of the bouquet and its resolution; (iii) the effect of telomere positioning at the bouquet on synapsis and chiasma formation.

#### MATERIALS AND METHODS

#### Plant Material

Seven wheat-rye introgression lines, each carrying one of the seven chromosome pairs of rye (S. cereale, 2n = 14) introgressed in hexaploid wheat T. aestivum, were used. The wheat-2R, wheat-3R, wheat-4R, wheat-5R, wheat-6R, and wheat-7R lines are disomic additions of the Imperial rye chromosome in a Chinese Spring wheat background (Driscoll and Sears, 1971). The wheat-1R line is a disomic substitution for chromosome 1A generated in hexaploid wheat Pavon 76 (Lukaszewski, 2008), and used for other purpose (Valenzuela et al., 2012). All wheat-rye introgression lines used were provided by A. J. Lukaszeski. The chromosome constitution of the plants studied was verified by FISH analysis of root tips in squashed preparations as described for meiosis. Seeds were germinated in November and grew in a greenhouse under natural light. Spikes at meiosis were cut and checked to establish the meiotic stage of each flower, One anther per flower was examined. When this anther was at prophase I or metaphase I the other 2 were fixed in 3:1 ethanol: acetic acid and stored at 4◦C.

### Fluorescence in Situ Hybridization

Preparations of fixed anthers were carried out as described (Maestra et al., 2002). Telomeres of wheat and rye chromosomes were labeled with the pAtT4 DNA probe, from Arabidopsis (Richards and Ausubel, 1988). The arrangement of telomere was used to identify the leptotene, zygotene, and pachytene stages as described (Corredor et al., 2007; Naranjo et al., 2010). Subtelomeric chromomeres of rye were visualized using clone pSc74, which contains a rye-specific 480-bp tandem repeat (Bedbrook et al., 1980; Cuadrado and Schwarzacher, 1998). The subtelomeric pSc74 signal identified the position of the adjacent rye telomere. Rye centromeres labeled with the rye-specific clone pAWRC.1 (Franki, 2001) were positioned at the bouquet and in the bivalents at pachytene and metaphase I. A fourth DNA probe, pUCM600, containing a rye-specific repeat (Gonzalez-Garcia et al., 2011) was added to paint each rye bivalent and quantify its level of synapsis in Pollen Mother Cells (PMCs) at pachytene. The probes, pUCM600, pAWRC.1, and pSc74, were used for the identification of each rye bivalent and the arms associated at metaphase I. Concentrations of DNA probes in the hybridization mixes were as described (Naranjo et al., 2010) In the analysis of the position of rye telomeres at the bouquet, probes pAtT4 and pAWRC.1 were labeled with biotin-16-dUTP and probe pSc74 with digoxigenin-11-dUTP. For painting of the rye bivalent at pachytene and metaphase I, the rye-specific DNA probes pAWRC.1 and pUCM600 were labeled with biotin-11-dUTP, while either digoxigenin-11-dUTP or biotin-16-dUTP were used in the case of probe pSc74. In nuclei at pachytene, the telomeres of wheat and rye chromosomes were visualized with the pAtT4 DNA probe labeled with digoxigenin-11-dUTP. Labeled probes were detected as described (Naranjo et al., 2010).

### Fluorescence Microscopy and Image Processing

Cells at mitotic metaphases and PMCs at pachytene and metaphase I were viewed under an Olympus BX60 fluorescence microscope equipped with an Olympus DP70 CCD camera. Cells at the bouquet stage were studied under an Olympus BX61 fluorescence microscope and processed as described (Naranjo, 2014)

## Chromosome Length and Telomere Arrangement

The length of rye chromosomes in both mitotic metaphases and meiocytes at pachytene, as well as different distances in nuclei at the bouquet stage and pachytene, were measured using the Adobe Photoshop CS4 software. In each wheat-rye addition line, the lengths of 10 mitotic chromosomes and 10 completely synapsed pachytene bivalents were scored. Separation between

the rye chromosome ends, and the major axis of each nucleus were measured in 2D projections of cells at the bouquet stage and pachytene. The distance of the ends of each rye chromosome to the center of the telomere cluster was also measured in cells at the bouquet. An average number of 100 cells per meiotic stage and line were scored.

### RESULTS

#### Rye Chromatin Remodeling During Leptotene Shows a Distinctive Pattern in the Wheat-4R Addition

Concomitant with the bouquet organization, chromatin undergoes a remodeling process that reduces its packaging level and causes a large elongation of chromosomes. At the bouquet, decondensation of rye chromatin was apparent but a considerable folding degree was still observed (**Figure 1**). This remnant folding of chromatin made it difficult to trace and measure the complete chromosome length. The assessment of chromatin reorganization in each rye chromosome was based on the variation of its length in completely synapsed bivalents at pachytene relative to that in mitotic metaphase. **Figure 2** compares the karyotype of rye chromosomes in both types of cells. In addition to the chromosome length, karyotypes show the position of the centromere and heterochromatin chromomeres. The two arms of all rye chromosomes show a distinctive pattern of heterochromatin markers. Chromosomes 4R and 5R bear a subtelomeric chromomere only in the short arm, the 4RL arm shows no marker and the subdistal chromomere of 5RL is smaller than that of 5RS. The subtelomeric chromomeres of the short and long arms of chromosomes 1R, 2R, 3R, 6R, and 7R differ in the heterochromatin contain. In addition to the subtelomeric marker, the arms 1RL, 2RS, 2RL, 6RL, and 7RL carry one or two intercalary chromomeres. Values of chromosome length and centromere index (100 × short arm length/chromosome length) are given in **Table 1**. The length of mitotic chromosomes ranges between 12.9 and 16.4 µm with an average of 15.1 µm. Bivalents at pachytene of chromosomes 1R, 2R, 3R, 5R, 6R, and 7R reached a length that ranges between six and seven times the size of the mitotic chromosome. Surprisingly, chromosome 4R is 11.2 times longer in pachytene than in mitotic metaphase. This result supports a different reorganization pattern of rye chromatin in leptotene in the wheat-4R addition relative to the others.

#### Clustering, but Not Dispersion, of Telomeres Is Chromosome Conformation-Dependent

The position of the telomere of the short arm of all rye chromosomes and of the long arms 1RL, 2RL, 3RL, 6RL, and 7RL in PMCs at early prophase I was identified by its close proximity to the adjacent subtelomeric heterochromatin marker. FISH analysis combining the telomeric and the rye-specific heterochromatin DNA probes in nuclei at the bouquet stage revealed whether rye telomeres were included in the telomere cluster or not. **Figure 3** shows nuclei with the two ends of

FIGURE 1 | Nucleus of the wheat-6R addition at the bouquet stage. Chromatin folding makes it difficult to trace the chromosomal axis along its entire length. Centromeres (C) and telomeres (tel) are indicated. Bar represents 10 µm.

chromosomes 2R and 3R, and the end of chromosome arm 4RS at the telomere cluster (**Figures 3A,B,D**, respectively). In all of these cases the homologous chromosome ends are intimately associated denoting the initiation of synapsis. The telomere of one chromosome arm 3RS, one chromosome arm 6RS and the two arms 4RS are not included in the telomere cluster in nuclei of **Figures 3C,E,F**, respectively. From the number of cells with zero, one or two homologous ends included in the telomere pole it was possible to estimate the probability of incorporation to the telomere cluster of the telomere of the seven short chromosome arms and the long arms 1RL, 2RL, 3RL, 6RL, and 7RL. This probability appears diagrammed in **Figure 4**. The telomere of both arms of metacentric chromosomes 2R, 3R, and 7R as well as the telomere of 1RL and 6RL migrated to the telomere pole in almost all cells. However, the telomere of the short arm of submetacentric chromosomes 4R, 5R, and 6R failed in its incorporation to the telomere pole in more than 25% of the meiocytes. On average, such telomeres were separated 10 µm from the telomere cluster. Accordingly, the probability of incorporation of the telomere of the short arm of the seven rye chromosomes and the centromere index were positively correlated (r = 0.828, t = 13.16, d.f. = 5, and p < 0.01). This result supports an effect of chromosome conformation in the positioning of telomeres during the organization of the bouquet. Although the centromere index of 1R identifies this chromosome as submetacentric, the location of the nucleolar organizing region on 1RS probably represents an additional factor conditioning its arrangement in leptotene. Consistent with the disturbed positioning of the telomeres of 4RS, 5RS, and 6RS is the reduction in the frequency of association of their subtelomeric markers in cells with the two homologous ends included in the telomere

TABLE 1 | Length (µm) of rye chromosomes in mitotic metaphase (M) and pachytene (P), P/M ratio and centromere index (100 × short arm length/chromosome length) in wheat-rye additions.

cluster. While the subtelomeric chromomeres of 4RS, 5RS, and 6RS were intimately associated on average in 46% of the cells studied, the homologous ends of metacentric chromosomes were in 86%.

The fact that telomere clustering was chromosome conformation dependent led to verify whether the dispersive telomere movement responsible of the bouquet disorganization was also affected by chromosome conformation or not. When both ends of rye chromosome pairs 1R, 2R, 3R, 6R, or 7R were included in the telomere cluster, they were situated at an average distance of 2 mµ. Such a distance is expected to increase because of telomere dispersion during zygotene. Thus, the spatial separation between the ends of each bivalent in nuclei at pachytene, in which the bouquet is completely disorganized, should provide an assessment of the effect of the dispersive telomere movement. The distance between the chromosome ends was scored in completely synapsed bivalents. A great between cells variation was found. **Figure 5** shows nuclei with the chromosomal ends of the rye bivalent relatively close or occupying diametrically opposed positions, respectively. The mean distance between the ends of each chromosome, as well as the size of the nuclear diameter, appear indicated in **Table 2**. The ends of metacentric chromosome 3R and submetacentric chromosomes 4R and 5R, show very similar separation and the same happens in the group formed by metacentric chromosomes 2R and 7R and submetacentric chromosome 6R. There is no significant correlation between the inter-chromosome ends distance and the centromeric index (r = 0.471, t = 1.194, d.f = 5, and p > 0.05). Thus, chromosome conformation does not affect the telomere movement during the bouquet dissolution. Between chromosomes variation in the degree of separation of the two ends was correlated to the size of the nuclear diameter (r = 0.858, t = 3.73, d.f. = 5, p < 0.05). Such a variation should at least in part be a result of manipulation during the realization of preparations. In fact, with the exception of chromosome 3R, the between ends distance/nuclear diameter ratio was similar in all remaining chromosomes. The telomere dispersion produced in zygotene separated the chromosomal ends to nuclear positions distant on average 43% of the nuclear diameter.

FIGURE 3 | 3D analysis of the arrangement of the subtelomeric chromomeres (green) of the short (S) and long (L) arm or rye chromosomes at the bouquet in wheat-rye additions. The telomere cluster (red) and the rye centromeres (c) are indicated. (A) Nucleus with the two ends of 2R at the telomere cluster. Homologous chromomeres of both arms are associated. (B) The subtelomeric chromomeres of 3RS and 3RL are associated and incorporated to the telomere cluster. (C) The subtelomeric chromomeres of 3RL are associated and incorporated to the telomere cluster. Only one 3RS telomere is in the telomere cluster. (D) The ends of 4RS are associated and locate at the telomere cluster. (E) The 4RS telomeres are separated and locate out of the telomere cluster. (F) Both ends of the chromosome pair 6R are separated, the two 6RL telomeres and one telomere of 6RS are included in the telomere cluster. Bars represent 10 µm.

## Rye Chromosome Arms Differ in the Level of Synapsis of Their Distal Half

In many plant species crossovers are non-random distributed along the chromosomes. In wheat, barley and rye, they are confined to the distal half of each chromosome arm (Lukaszewski and Curtis, 1993; Akhunov et al., 2003; Lukaszewski, 2008; Valenzuela et al., 2012; Higgins et al., 2014). Such regions should undergo synapsis in order to facilitate the occurrence of crossovers. Most rye pachytene bivalents showed synapsed the distal half of both chromosome arms (**Figures 2C,E**, **5A,B**), however, unmatched chromosome arms were also observed (**Figures 6A,B**). Failure of synapsis in the distal half varied between chromosomes arms (**Figure 6C** and **Table 3**). Chromosome arms 5RS and

6RS, with disturbed telomere migration during leptotene, showed the lowest level of synapsis. Chromosome arm 4RS, despite restricted telomere migration, completed synapsis in most PMCs. Because synapsis progresses from the end to the center of the chromosomes (Corredor et al., 2007), unsynapsed stretches were more often observed in proximal regions, especially in chromosomes 6R and 7R (**Table 3**).

TABLE 2 | Mean separation between the two ends of each completely synapsed rye bivalent (S-L distance), average nuclear diameter (ND) and S-L distance/ND rate in PMCs at pachytene of wheat-rye additions.

### Chiasma Formation Is Affected in Arms That Fail to Synapse and in Arms With a High Level of Synapsis

Chiasmata formed by rye chromosomes were inferred from the occurrence of association between each homologous arm pair in PMCs at metaphase I (**Figure 7**). Two homologous arms bound at metaphase I had formed at least one chiasmata, but it is difficult to establish whether additional chiasmata were present or not. Thus, the frequency of homologous arms association at metaphase I represents an underestimation of the number of chiasmata per chromosome arm. **Table 4** shows the frequency of association for the different rye chromosome arms as well as the total number of associations per cell. Both arms of chromosomes 1R, 3R, and 7R, and the long arm of chromosome 2R formed chiasmata with frequencies higher than 90%. The remaining arms showed lower frequencies, especially the arms 2RS, 4RS, 4RL, 5RS, and 6RS, whose frequencies ranged between 25 and 70%. The total number of associations varied between lines. A maximum number of 42 arm associations can be formed in the wheat-1R line and 44 in the

FIGURE 5 | Variable separation of the ends of the rye bivalent in pachytene nuclei of the wheat-3R addition. (A) Chromosome ends closely located. (B) Very distant chromosome ends. Bars represent 10 µm.

other six lines. Thus, chiasma formation failed not only between rye chromosome arms but also between wheat chromosomes, especially in the addition lines wheat-4R and wheat-5R.

#### DISCUSSION

The majority of rye chromosomes of the addition lines studied undergo a meiotic behavior that it is different from that observed in rye itself. This is apparent, if attention is paid to the number of chiasmata formed. In rye, a total of 13.4 (1.9/chromosome) chiasmate bonds are formed at metaphase I (Naranjo and Lacadena, 1980). This figure represents an association frequency of 95.7% per chromosome arm. In the wheat-rye additions, the arms 2RS, 4RS, 4RL, 5RS, and 6RS show much lower frequencies of association (<71%, **Table 4**). But let us analyze separately the different steps of prophase I that precedes chiasma formation.

#### Chromatin Remodeling

Remodeling of chromatin produced in leptotene leads to a more relaxed chromosome organization, which most likely facilitates the search of the homologous partner. During the SC assembly, the axial elements (chromosomes) become shortened. This shortening has been quantified in electron microscopic studies of spread nuclei of both rye (Gillies, 1985) and wheat (Martínez et al., 2001). The length of SCs at pachytene is approximately 80% of the length in zygotene. One can assume that rye chromosomes suffered a similar shortening in the course of zygotene in wheatrye addition lines. According to the length of pachytene bivalents in the wheat-rye additions studied, rye chromosomes fall into two categories, one corresponds to chromosome 4R, with a length of 172.8 µm, and the other contains the six remaining chromosomes, with an average length of 100.6 µm. Such a divergence does not exist in rye itself. The length of SC bivalents at pachytene in two different stocks of rye, ranged between 72.2– 61.8 µm and 64.0–54.1 µm, respectively (Gillies, 1985). Thus, the appearance of two different chromatin reorganization patterns between the chromosomes of rye suggests chromosome-specific responses to the new genetic background in which they have been introgressed.

One could assume that differences in length between chromosomes in pachytene might be a result of differences in their DNA content, given the large size, 8.1 Gb, of the rye genome (Doležel et al., 1998). Chromosome 4R consists of a DNA sequence of 1435 Mb, which represents 17.4% of the rye genome. This sequence is higher than those of the other six chromosomes, which range between 1023 Mb (1R) and 1253 Mb (2R) with an average size of 1136 Mb/chromosome (Martis et al., 2013). Taken into account the chromosomal length, the group of six chromosomes shows a mean packaging level of 11.3 Mb per µm in the pachytene bivalents while the ratio decreases to 8.3 Mb per µm in the case of 4R. The packaging level of chromosome 4R represents 73% of that observed in the other chromosomes. Thus, differences between chromosomes in their length at pachytene are the result, not only of differences in the DNA content, but also of distinct patterns of chromatin reorganization during early prophase I.

TABLE 3 | Frequency (%) of PMCs with synapsis involving the distal half or the proximal half of individual rye chromosome arms at pachytene in disomic wheat-rye additions.

It is also noteworthy that the mean length of rye chromosomes at pachytene is much higher in the addition lines, 110.9 µm, than in rye itself, 62.5 µm (Gillies, 1985). The mean length of 110.9 µm found in the wheat-rye additions fits the average length of 90.2 µm of wheat chromosomes (Martínez et al., 2001). This variation of the mean chromosome length supports that the pattern of chromatin remodeling of a given chromosome at leptotene is controlled by the genetic background in which such a chromosome is present. In pachytene bivalents, the two homologues are closely juxtaposed along their entire length by the installation of the SC. Each chromatid of the bivalent consists of a linear arrangement of loops whose bases are anchored to the SC lateral element. Axes of sister chromatids are closely juxtaposed and their loops project out of the SC (Zickler and Kleckner, 2015). The density of loops along the pachytene axis is quite conserved in different organisms (∼20 loops per micron) (Kleckner, 2006). The evolutionarily conserved loop spacing implies that variation in the chromosome axis length should be accompanied by inversely correlated differences in the loops size. This is manifested in mice mutants that have altered the meioticspecific cohesin Smc1β and the SC lateral component Sycp3; relatively long chromosome axes project short loops while shorter chromosome axes are coupled with longer loops (Revenkova and Jessberger, 2006; Novak et al., 2008). Thus, the increase of the length of rye pachytene bivalents in wheat-rye addition lines relative to plants of rye, as well as differences between lines, should be the result of modifications in the number and size of the loops.

Little is known about the impact of molecular interactions underlying the introgression of rye chromatin in a wheat background. A comparison of the transcriptomes of wheat, barley and a wheat-barley ditelosomic 7HL addition revealed reprograming of the transcriptomes of wheat and 7HL in the addition line (Rey et al., 2018). Only 3% of wheat genes underwent a different transcription rate in the addition line relative to wheat, while 42% of genes of 7HL were up- or

TABLE 4 | Frequency (%) of association of individual rye chromosomes arms and total number of bounds in cells at metaphase I of disomic wheat-rye additions.

aAfter Naranjo (2014).

down-regulated in the addition line relative to barley. These results evidence the existence of interactions between genes of both species, which may change with the introgressed chromosome. Thus, chromosome-specific molecular interactions affecting genetic functions related to the reorganization of chromatin during prophase I could cause differences between lines. Whether comparable genetic interactions exist or not in other sets of wheat-rye additions is unknown. This point may be addressed in future research works.

#### Clustering and Dispersion of Telomeres

Telomeres of the long arms 1RL, 2RL, 3RL, 6RL, and 7RL as well as telomeres of the short arm of metacentric chromosomes 2R, 3R, and 7R, were included in the telomere cluster in almost all PMCs. By contrast, the telomere of the short arm of submetacentric chromosomes 4R, 5R, and 6R showed a lower probability of inclusion in the telomere cluster. This result reinforces previous studies supporting a chromosome conformation dependence in the ability of telomeres to converge in the telomere pole (Naranjo et al., 2010; Naranjo, 2014). Chromosome 1R with a centromeric index lower than that of 4R shows, however, a behavior more similar to that of metacentric chromosomes. It is possible that the presence in its short arm of the nucleolar organizing region, where chromatin is more relaxed, could affect the arrangement of this chromosome during leptotene.

The organization of the bouquet is the result of three interdependent events: the attachment of telomeres to the cytoskeleton through a nuclear envelope protein bridge, the oriented telomere cytoskeleton-mediated movement, and the clustering of telomeres in a small region of the nuclear membrane. Some of these steps were affected in the telomeres of the arms 4RS, 5RS, and 6RS. The link between the cytoskeleton and the chromosome ends during meiosis is provided by a complex of proteins among which are the transmembrane SUN (sad1/UNC-84) domain protein that interact with the chromosome ends (Starr, 2009) and the KASH (Klarsicht/Anc/Syne homology) domain protein that in turn interact with elements of the cytoskeleton (Starr and Fridolfsson, 2010). The function that the SUN and KASH proteins develop in fungi and animals is conserved in the plant kingdom from the most ancestral plant species to advanced angiosperms (Poulet et al., 2017). In maize and Arabidobsis, SUN protein foci spread through the nuclear envelop during leptotene, cluster in the periphery of one hemisphere at the bouquet, and back to spread through all the nuclear envelop during and beyond pachytene (Murphy et al., 2014; Varas et al., 2015). This SUN proteins distribution suggests that any chromosome end, regardless its location, has the possibility of finding its attachment to the nuclear envelope prior to migration. Thus, failure of the attachment of telomeres of 4RS, 5RS, and 6RS to the nuclear envelope does not seem the reason of their disturbed positioning.

Another explanation of the behavior of telomeres of 4RS, 5RS, and 6RS is that they have to cover longer distances than other telomeres to reach the telomere pole during the bouquet organization. This explanation is based under the assumption that the site of the telomere pole is most likely installed in the region of the telomeric hemisphere with the highest density of telomeres, that is, in the region where telomeres of long arms are located. In such a situation, telomeres of metacentric chromosomes can be equidistant relative to the telomere pole, while telomeres of the short arms of submetacentric chromosomes lay farther away of this pole. In support of this assumption are two facts: (i) telomeres of the long arms are included in the telomere cluster in almost all cells, (ii) the centromeric end of telocentric chromosomes fails also in its incorporation to the telomere pole (Corredor and Naranjo, 2007). The telomere of the centromeric end of the 5RL telocentric locates at the centromere pole at early leptotene. In the course of leptotene this chromosome end abandons the centromere pole to cluster with the other telomeres in the opposite pole. However, only 51% of the centromeric ends incorporate into the telomere cluster at the consolidated bouquet stage.

The effect of chromosome conformation on telomere clustering is not extended to the movement produced during zygotene, which disperses telomeres through all the nuclear periphery. Telomeres of any chromosome pair are very close at the bouquet and their separation seems to be at random, i.e., without any programed itinerary. Within lines variation of the distance between the ends of the rye bivalent at pachytene agrees with a random dispersion of telomeres. Most likely, the overall arrangement of bivalents in the nucleus and the simultaneous movement of all chromosomes should condition severely the distance covered by each telomere.

#### Synapsis and Chiasma Formation

Restriction of chiasma formation to the distal half of chromosome arms in species such as wheat, rye or barley (Lukaszewski and Curtis, 1993; Akhunov et al., 2003; Lukaszewski, 2008; Valenzuela et al., 2012; Higgins et al., 2014) implies that such chromosome regions should develop synapse regularly. This was so for most rye chromosome arms. All of the arms with normal telomere migration during the bouquet organization showed normal synapsis. Failure of synapsis affected mainly the short arm of submetacentric chromosomes 5R and 6R. Synapsis failure in these arms was preceded of disturbed migration of their telomere during the bouquet formation, which most likely impeded the occurrence of physical interactions and synapsis initiation between their subtelomeric homologous. The behavior of 5RS and 6RS is in

contrast with that of 4RS, which completed synapsis despite its telomere failed also to be included in the telomere cluster. What makes chromosome arm 4RS different? This arm reaches a much higher length at pachytene than 5RS and 6RS. Unfolding of chromatin produced in leptotene obliges chromosomes to move and span the entire nucleus because their length exceeds largely the nuclear diameter. The intensity of the movement is expected to increase with the length reached by each chromosome. Such a chromosome dynamics might promote the occurrence of chance encounters between intercalary homologous regions regardless their respective telomeres were separated. Under this assumption, the probability of intercalary interactions between homologues is expected to be higher in chromosome arm 4RS than in 5RS and 6RS. Synapsis initiated intercalarily in 4RS could extend later to its distal region, although the telomeres were not initially associated. This situation is comparable to that found in an inversion heterozygote for the long arm of chromosome 1R. In this heterozygote, the crossover-rich region is positioned distally in the standard chromosome and proximally in the inverted chromosome at the initiation of meiosis, i.e., they are located in opposite poles of the nucleus. However, during zygotene, a non-telomere led chromosome movement, the movement generated by chromosome elongation, makes it possible that these homologous regions find one another and that the entire arm complete synapsis (Lukaszewski, 2008; Valenzuela et al., 2012; Naranjo, 2014). Thus, the high level of synapsis of chromosome 4R can be the result of homologous interactions facilitated either by the bouquet organization or, in cells with disturbed migration of the 4RS telomere, by the chromosome movement derived of chromatin unfolding. The distal half of the short arm of metacentric chromosome 7R showed a level of synapsis of 81%, which is higher than that of 5RS (73%) or 6RS (57%) but lower than the level of the remaining arms (**Table 2**). However, the frequency 92% of association of chromosome arm 7RS at metaphase I suggests an almost normal level of synapsis in the anther where metaphase I was studied.

Chiasma distribution in rye chromosomes of wheat-rye addition lines was earlier studied (Drögenmüller and Lelley, 1984). However, the number of chiasmate bonds per bivalent instead the association frequency of each chromosome arm was reported. Nevertheless, chromosomes 5R and 6R formed a more reduced number of chiasmata than the remaining rye chromosomes. Reduction of the number of chiasmata in chromosomes 5R and 6R affects mainly their short arm

#### REFERENCES

(**Table 4**) and is the consequence of the synapsis failure that such arms showed at pachytene. However, other arms with normal synapsis suffered a considerable reduction of the chiasma frequency. This was the case of the arms 2RS, 4RS, and 4RL. The wheat-4R addition line showed the lowest number of chiasmate bonds, 37.9, which is far from 44, the expected maximum number. Environmental variation could explain minor differences between lines, but the low chiasma frequency observed in the wheat-4R addition suggests a negative effect of the genetic background of this line on chiasma formation, which affects both wheat and rye chromosomes. Addition of individual chromosomes of rye to wheat produce changes in traits such as spike and spikelet morphology (Driscoll and Sears, 1971). Addition of chromosome 4R increases also the length of the anthers and the number of pollen grains, while the addition of 1R or 2R reduce fertility (Nguyen et al., 2015). It is possible that chromosome 4R carries some genetic information that interferes on chiasma formation. The wheat-2R addition forms, however, a high number of chiasmata, 41.3. The reduction observed in chromosome arm 2RS seems to be chromosomespecific and derived from the genetic constitution or chromatin organization of this arm. In fact, chromosome arm 2RS carries a large subtelomeric heterochromatin chromomere and such a heterochromatin has been shown to reduce the number of chiasmata of rye chromosomes in wheat-rye derivatives (Naranjo and Lacadena, 1980).

#### AUTHOR CONTRIBUTIONS

TN designed and conducted the research and wrote the work.

#### FUNDING

This work was supported by grant AGL2015-67349-P from Dirección General de Investigación Científica y Técnica, Ministerio de Economía y Competitividad of Spain.

#### ACKNOWLEDGMENTS

I would like to thank A. Lukaszewski for kindly supplying the wheat-rye introgression lines.

synapsis during the telomere-clustering (bouquet) stage of meiotic prophase. J. Cell Sci. 113, 1033–1042.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Naranjo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-09-00880 June 28, 2018 Time: 17:56 # 13

# Homoeologous Chromosomes From Two *Hordeum* Species Can Recognize and Associate During Meiosis in Wheat in the Presence of the *Ph1* Locus

María C. Calderón1 , María-Dolores Rey 2 , Antonio Martín1 and Pilar Prieto1 \*

1 Plant Breeding Department, Institute for Sustainable Agriculture, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Córdoba, Spain, 2 John Innes Centre, Norwich Research Park, Norwich, United Kingdom

#### *Edited by:*

Mónica Pradillo, Complutense University of Madrid, Spain

#### *Reviewed by:*

Eric Jenczewski, INRA Centre Versailles-Grignon, France Pierre Sourdille, INRA Centre Auvergne Rhône Alpes, France

> *\*Correspondence:* Pilar Prieto<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

*Received:* 13 February 2018 *Accepted:* 13 April 2018 *Published:* 01 May 2018

#### *Citation:*

Calderón MC, Rey M-D, Martín A and Prieto P (2018) Homoeologous Chromosomes From Two Hordeum Species Can Recognize and Associate During Meiosis in Wheat in the Presence of the Ph1 Locus. Front. Plant Sci. 9:585. doi: 10.3389/fpls.2018.00585 Understanding the system of a basic eukaryotic cellular mechanism like meiosis is of fundamental importance in plant biology. Moreover, it is also of great strategic interest in plant breeding since unzipping the mechanism of chromosome specificity/pairing during meiosis will allow its manipulation to introduce genetic variability from related species into a crop. The success of meiosis in a polyploid like wheat strongly depends on regular pairing of homologous (identical) chromosomes and recombination, processes mainly controlled by the Ph1 locus. This means that pairing and recombination of related chromosomes rarely occur in the presence of this locus, making difficult wheat breeding trough the incorporation of genetic variability from related species. In this work, we show that wild and cultivated barley chromosomes associate in the wheat background even in the presence of the Ph1 locus. We have developed double monosomic wheat lines carrying two chromosomes from two barley species for the same and different homoeology chromosome group, respectively. Genetic in situ hybridization revealed that homoeologous Hordeum chromosomes recognize each other and pair during early meiosis in wheat. However, crossing over does not occur at any time and they remained always as univalents during meiosis metaphase I. Our results suggest that the Ph1 locus does not prevent chromosome recognition and pairing but crossing over between homoeologous. The role of subtelomeres in chromosome recognition is also discussed.

Keywords: wheat, barley, homoeologous pairing, introgressions, meiosis, chromosome recognition

## INTRODUCTION

More than two-thirds of global cropland features annual grain crops, which represent roughly 70% of humanity's food energy needs and typically grown in monoculture. Annual grain production, at its current scale, is fundamentally unsustainable. Thus, the growing human population demands greater crops, more productive and better adapted to specific agro-climatic conditions (Godfray et al., 2010). Plant breeders are playing a major role in worldwide efforts to understand gene functions and interactions with the aim of increasing quality and productivity of major crops. Wide-crossing in plant breeding is an important tool and sometimes the results are the starting point for new crops (Omara, 1953). For example, wide-crossing has been carried out in the Triticeae tribe, which includes wheat, to develop new plant species such as ×Triticosecale, obtained after crossing wheat and rye, or ×Tritordeum, an amphyploid between the wild barley Hordeum chilense Roem. et Schult. and wheat (Omara, 1953; Martín and Sanchez-Monge Laguna, 1982). Breeders have also used related species as genetic donors for widening the genetic basis of wheat to get for example wheat cultivars better adapted to specific agro-climatic conditions, improving the quality or carrying resistance to diseases (Lukaszewski, 2000; Liu et al., 2005; Calderón et al., 2012; Rey et al., 2015a). In fact, there are many wild species carrying interesting traits that would be useful to be exploited in wheat breeding programmes, but unfortunately, hybridization between wheat and a wild related species produces only a low level of chromosome pairing and recombination. So understanding wheat genetics and genome organization is essential for plant breeding purposes.

Bread wheat is an hexaploid, which possesses three sets of related chromosomes because of doubling of chromosomes following sexual hybridization between closely related species. However, chromosomes associate regularly in pairs in wheat during meiosis, the cellular process to produce gametes in sexually reproducing organisms. Thus, at meiosis each chromosome only recognize and associate with its homologous and not with the related (homoeologous) chromosomes, which have a similar gene content and order but differ in repetitive DNA sequences. Several pairing homologous (Ph) genes control chromosome associations in wheat, although the major effect is due to the Ph1 locus (Sears, 1976). The efficiency of chromosome associations during meiosis have a big influence on the fertility of wheat plants, being crucial for success in breeding, but has a negative effect preventing pairing and recombination between wheat chromosomes and those from related species. Therefore, it seems reasonable to go deeper into the knowledge of the biology of chromosome associations during meiosis in wheat, which will be valuable for wheat breeding.

Chromosome dynamics during meiosis have been extensively studied in a polyploidy such as hexaploid wheat (Moore, 2002; Corredor et al., 2007; Colas et al., 2008; Naranjo and Corredor, 2008). It is now well established that both interactions during recombination at the DNA level and assembly of a meiosisspecific proteinaceous structure known as the synaptonemal complex (SC) play roles in stabilizing associations between homologous chromosomes. However, how homologs became colocalized and how initial recognition is accomplished to establish chromosome associations remains poorly understood. When a chromosome recognizes its homolog (and not another chromosome) in wheat, a localized conformational change in adjacent chromatin is triggered in both partners. This process facilitates recognition and association of homologous versus homoeologous chromosomes and is affected by the Ph1 locus (Prieto et al., 2005; Greer et al., 2012). Thus, Ph1 stabilizes wheat during meiosis by both, promoting homolog synapsis during early meiosis and preventing homoeologous recombination later in meiosis (Martín et al., 2014, 2017). The effect on synapsis occurs during the telomere bouquet Ph1 stage, when promotes more efficient homologous synapsis, thereby reducing the chance of homoeologous synapsis (Martín et al., 2017). The effect on CO formation occurs later in meiosis, when Ph1 prevents MLH1 sites (Double Holliday Junctions marked to become COs) on synapsed homoeologues from becoming COs. In addition, it has been also described that the level of a ZIP4 paralog included within the Ph1 locus alters the number of CO between homoeologous chromosomes (Rey et al., 2017).

Efforts focused on centromeres and telomeres behavior during meiosis have been also made (Martinez-Perez et al., 2000, 2001, 2003; Naranjo et al., 2005). Telomeres, which are highly conserved structures among plants, including wheat (Simpson et al., 1990; Ganal et al., 1991; Schwarzacher and Heslop-Harrison, 1991), play an important role on initial chromosome associations at the onset of meiosis. In this stage, the association of telomeres in a bouquet facilitates the search and recognition of homologous chromosomes by bringing chromosomes closer (Corredor and Naranjo, 2007; Koszul et al., 2008; Moore and Shaw, 2009) and its formation is affected by the Ph1 locus (Richards et al., 2012). Subtelomeres, which are the telomere associated sequences (TAS), are highly polymorphic and extraordinarily dynamic sequences (Eichler and Sankoff, 2003). The complex and variable nature of subtelomeres has made difficult to assess the possible functions(s) of these regions so far, but studies on Arabidopsis and Hordeum subtelomeres might suggest a possible role on chromosome specificity between homolog chromosomes at the onset of meiosis (Kotani et al., 1999; Heacock et al., 2004; Calderón et al., 2014). In fact, subtelomeres in Hordeum showed high variability not only from different chromosomes but also among chromosome arms within the same chromosome (Schubert et al., 1998; Prieto et al., 2004b). Thus, the copy number of the subtelomeric HvT01 sequence was variable among chromosomes in both H. chilense and H. vulgare. Since chromosome associations are initiated at the distal regions of the chromosomes and homologous chromosomes are zipping up from those to the centromeres (Prieto et al., 2004a; Corredor et al., 2007), it seems reasonable to go deeper into the role of the subtelomeric regions on homolog chromosome associations, rather than focusing on features that are common to all chromosomes like telomeres.

The addition of a pair of "alien" chromosomes to the full genome complement of a crop species is commonly used as a first step for accessing genetic variation from the secondary gene pool, but addition lines are also relevant for understanding meiotic pairing behavior and chromosome structure (Friebe et al., 2005; Lukaszewski, 2010). Sets of both cultivated (Hordeum vulgare) and wild (H. chilense) barley addition lines in a hexaploid wheat background were developed (Islam et al., 1978, 1981; Miller et al., 1982) and have potential in plant meiosis studies. Certainly, it allows tracking one specific pair of chromosomes or chromosome segments within the wheat background using genomic in situ hybridization (GISH) and study chromosome rearrangements and associations exclusively in a pair of homologs (Naranjo et al., 2010; Rey et al., 2015b).

In this study, we have developed double monosomic addition lines of wild and cultivated barley in wheat for the same and for different homoeology group to go deeper into the knowledge of chromosome associations during meiosis. These double monosomic addition lines enabled to distinguish chromosomes from two different barley species in the wheat background, observe conformational changes during meiosis and analyze whether subtelomeres might play a role on chromosome recognition/pairing at early meiosis in the absence of homologs. Results showed that homoeologous chromosomes can recognize each other to associate correctly in pairs, even in the presence of the Ph1 locus, although crossing over does not occur as they remained as univalents during metaphase I.

### MATERIALS AND METHODS

### Plant Material

Crosses between H. chilense and H. vulgare addition lines in bread wheat (Triticum aestivum cv. Chinese Spring; AABBDD + pair of HchHch and AABBDD + pair of HvHv , respectively) for the same and for different homoeology group were made to obtain double monosomic wheat lines carrying one H. chilense and one H. vulgare chromosome for the same and for different homoeology group. H. chilense and H. vulgare addition lines were kindly provided by Steve Reader, JIC, Norwich, UK. The presence of each Hordeum sp. chromosome in parental and F1 wheat lines used in this work was confirmed by both PCR assays previously described (Liu et al., 1996; Hagras et al., 2005) and in situ hybridization.

Seeds obtained from genetic crosses were germinated on wet filter paper in the dark for 5 days at 4◦C, followed by a period of 24h at 25◦C. Plants were then growth in the greenhouse at 26◦C during the day and 18◦C during the night (16 h photoperiod).

### Fluorescence *in Situ* Hybridization

Fluorescence genomic in situ hybridization (GISH) was used to study chromosome associations between H. chilense and H. vulgare chromosomes in the wheat background as described previously (Prieto et al., 2004b). Root tips were collected from germinating seeds and were pre-treated for 4 h in a 0.05% colchicine solution at 25◦C and fixed in 100% ethanol-acetic acid, 3:1 (v/v), for at least a week at room temperature. Spikes in meiosis were collected from mature plants and preserved in 100% ethanol-acetic acid, 3:1 (v/v) until were used to characterize chromosome associations. Chromosome spreads were prepared from both root tips cells and pollen mother cells (PMCs) at meiosis. Root tips and anthers were macerated in a drop of 45% glacial acetic acid on ethanol-cleaned slides, squashed under a cover slip and dipped in liquid nitrogen in order to fix the plant material on the slide. The cover slip was removed and the slides were air-dried and stored at 4◦C until used.

Both total genomic H. vulgare and H. chilense DNA were labeled by nick translation with biotin-11-(Boehringer Mannheim Biochemicals, Germany) and digoxigenin-11-dUTP (Roche Applied Science, Indianapolis, IN, USA), respectively, and used as probes. Both probes were mixed to a final concentration of 5 ng/µl in the hybridization mixture. The hybridization mixture consisted of 50% formamide, 2 × SCC, 5 ng of biotin-labeled or digoxigenin-labeled probe, 10% dextran sulfate, 0.14 µg of yeast tRNA, 0.1 µg of sonicated salmon sperm DNA and 0.005 µg of glycogen. Biotin-labeled H. vulgare DNA and digoxigenin-labeled H. chilense DNA were detected with a streptavidin- Cy3 conjugate (Sigma, St. Louis, MO, USA) and antidigoxigenin-FITC (Roche Diagnostics, Meylan, France), respectively. Chromosomes were counterstained with DAPI (4′ , 6-diamidino-2-phenylindole) and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA).

Chromosome spreads from somatic cells and anthers of the F1 wheat lines were reprobed with the barley subtelomeric tandem repeat HvT01, which was obtained by amplification by the polymerase chain reaction (PCR) from genomic DNA from the barley cv. Betzes using primers made according to the published sequence (Belostotsky and Ananiev, 1990). PCR conditions were previously described by Prieto et al. (2004b). The PCR product corresponding to this barley satellite HvT01 probe was labeled with digoxigenin-11-dUTP, (Roche Applied Science, Indianapolis, IN, USA) by nick translation and detected with antidigoxigenin-FITC (Roche Diagnostics, Meylan, France). Meiosis metaphase samples were also reprobed to label centromeres using the RT sequence included in the barley centromeric BAC7 (Hudakova et al., 2001), amplified by PCR following the same conditions as the amplification of the CCS1 centromeric repeat (Aragón-Alcaide et al., 1996), labeled with biotin-11-(Boehringer Mannheim Biochemicals, Germany) and detected with the streptavidin- Cy3 conjugate (Sigma, St. Louis, MO, USA).

#### Fluorescence Microscopy and Image Processing

Hybridization signals were visualized using a Nikon Eclipse 80i epifluorescence microscope. Images were captured with a Nikon CCD camera using the Nikon 3.0 software (Nikon Instruments Europe BV, Amstelveen, The Netherlands) and processed with Photoshop 11.0.2 software (Adobe Systems Inc., San Jose, California, USA).

### Statistical Analysis

Statistical analyses were performed using STATISTIX 10.0 software (Analytical Software, Tallahassee, FL, USA). Anaphase I combinations were evaluated by an analysis of variance (ANOVA) as a completely randomized design. This analysis included a tangent transformation in the anaphase I combination where only one pole of the meiocytes showed H. chilense and H. vulgare signals. Tetrad combinations were analyzed by the Kruskal–Wallis test (nonparametric one-way analysis of variance).

## RESULTS

#### Development of Double Monosomic *H. vulgare-H*. Chilense Addition Lines in Wheat

Crosses between disomic H. chilense and H. vulgare addition lines in bread wheat carrying chromosomes 7Hch and 7Hv , respectively, were made to obtain double monosomic barley additions in wheat carrying homoeologous H. chilense and H. vulgare chromosome 7. Similarly, genetic crosses between disomic H. chilense and H. vulgare addition lines in bread wheat for chromosomes 7Hch and 5Hv , respectively, were made to obtain double monosomic wheat lines carrying non-homoeologous chromosomes 7Hch and 5Hv . Finally, to corroborate observations on chromosome associations in a different homoeology group, we also developed genetic crosses between disomic H. chilense and H. vulgare addition lines in bread wheat for chromosomes 5Hch and 5Hv , respectively, to obtain double monosomic barley additions in wheat lines carrying homoeologous H. chilense and H. vulgare chromosomes for group 5. The F1 hybrid progeny from each genetic cross was analyzed by GISH to ensure that they retained the expected both H. chilense and H. vulgare chromosomes (**Figure 1**). All the plants from all the genetic crosses carried both barley chromosomes. In addition, fluorescence in situ hybridization (FISH) was also performed in these wheat lines using the barley subtelomeric HvT01 repeat as a probe to label polymorphisms between the subtelomeric regions from the H. chilense and H. vulgare chromosomes added to the wheat background (**Figure 1**). Chromosome 7Hv had two strong signals for the barley subtelomeric HvT01 sequence on both chromosome arms

meanwhile there was only a weaker signal on the short arm of chromosome 7Hch. Both 5Hch and 5Hv chromosomes had a signal on the short arm for the HvT01 probe, which was stronger in the case of 5Hv chromosome, and only a weak signal on the subtelomeric region of the long arm of chromosome 5Hv was detected, which sometimes cannot be clearly seen and depended on the FISH experiment (**Figure 1**). No HvT01 subtelomeric signals were detected on the wheat chromosomes. The F1 progeny from each genetic cross was also growth until meiosis with the aim of studying the meiotic behavior of both Hordeum chromosomes by in situ hybridization in PMCs in the wheat background.

### Homoeologous Wild and Cultivated Barley Chromosomes Can Fully Associate in Pairs in Wheat in the Presence of the Ph1 Locus

Chromosome pairing was analyzed during early meiosis by GISH in F1 plants carrying one copy of H. chilense and one copy of H. vulgare homoeologous chromosomes (7Hch and 7Hv ) and it was compared to those carrying non-homoeologous

H. chilense and H. vulgare chromosomes (7Hch and 5Hv , respectively). Experiments were developed in around a 100 cells of each genomic combination in prophase I of meiosis. Both wild and cultivated barley chromosomes were visualized simultaneously in the wheat background (**Figure 2**). In both cases, H. chilense and H. vulgare chromosomes were in proximity in the nucleus in early prophase (**Figures 2a,c**). As meiosis progressed, GISH experiments showed homoeologous H. chilense and H. vulgare chromosomes always fully-associated in pairs along the whole chromosome (**Figure 2b**). In contrast, nonhomoeologous Hordeum chromosomes 7Hch and 5Hv were not observed associated at this meiotic stage at any time, remaining always un-associated (**Figure 2d**).

GISH experiments were also carried out in cells in prophase I of meiosis in F1 plants carrying homoeologous chromosomes from H. chilense and H. vulgare for another homoeology group (group 5), with the aim of confirming the observations on chromosome associations between homoeologous chromosomes 7Hch and 7Hv in the wheat background. Results showed that homoeologous Hordeum chromosomes 5Hch and 5Hv did also associate in pairs during early meiosis in the wheat background, even in the presence of the Ph1 locus (**Figures 2e,f**), suggesting that chromosome pairing between homoeologous chromosomes from two different Hordeum species is not hampered by the Ph1 locus. In addition, results suggested that homoeologous barley chromosomes shared enough similar DNA sequences to

recognize each other, a conformational chromatin change is observed in both homoeologues and chromosomes are finally associated completely in pairs.

### Subtelomeres Might Hamper Chromosome Associations Between Non-homologous Hordeum Chromosomes in the Wheat Background

We have described that in the absence of homologous chromosomes, wild and cultivated barley homoeologous chromosomes can still recognize each other and associate completely in pairs during early meiosis. In addition, we have observed that, in these cases, initial chromosome recognition occurred by the chromosome ends where none or little copy number of the subtelomeric HvT01 repeat were detected, i.e., the long arm of homoeologous chromosomes 5Hch and 5Hv (**Figure 3**). Thus, homoeologous Hordeum chromosomes did recognize and associate in pairs by these chromosome ends, which made chromosome recognition less restrictive and allowed homoeologous to recognize and associate in pairs. These observations were similar in the cells detected at the same stage from the double monosomic 7Hch7Hv addition line in wheat (data not shown). Moreover, the observations were consistent in all the cells detected at the initiation of pairing (26 and 18 cells from the double monosomic 5Hch5Hv and 7Hch7Hv addition lines in wheat, respectively). Once homoeologous chromosomes had associated by these chromosome ends, a conformational change was observed along both homoeologous Hordeum chromosomes and pairing progressed along both chromosomes allowing a complete chromosome association between them (**Figure 3**).

### Crossing Over Does Not Occur Between Wild and Cultivated Barley Chromosomes in Wheat Although They Previously Associated in Early Meiosis

Once we observed full chromosome associations between homoeologous Hordeum chromosomes during early meiosis in the wheat background, we also analyzed chromosome behavior of both wild and cultivated barley chromosomes during metaphase I of meiosis in PMCs from double monosomic H. chilense and H. vulgare additions in wheat lines, both for the same and different homoeologous chromosomes. Meiosis metaphase I was also checked in the disomic H. chilense and H. vulgare addition lines in wheat used as parental lines for the genetic crosses developed in this work, to obtain the double H. chilense and H. vulgare double monosomic addition lines. Chromosome stability of the parental lines was confirmed as the homologous barley chromosomes carried in the H. chilense and H. vulgare disomic addition lines were always observed associated in pairs, indicating that crossing over occurred between homologous barley chromosomes (**Figure 4**). Similarly, wheat chromosomes associated correctly in bivalents at meiosis metaphase I and orientated by centromeres properly in double monosomic H. chilense and H. vulgare additions in wheat lines, both for the same and different homoeologous chromosomes (**Figure 5**). In contrast, H. chilense and H. vulgare chromosomes remained always un-associated in all the cells analyzed for the three different genetic combinations analyzed (**Figure 5**), despite the fact that homoeologous H. chilense and H. vulgare chromosomes (7Hch7Hv and 5Hch5Hv , respectively) did completely associate in pairs in early meiosis. These observations suggested that, although wild and cultivated barley homoeologous chromosomes can fully associate during pachytene, crossing over did not occur later between these chromosomes. Consequently, Hordeum homoeologous chromosomes were never observed associated by chiasmata during metaphase I and always remained as univalent (**Figure 5**), suggesting other requirements for crossing over rather than full previous chromosome associations or similarities in the DNA sequence.

#### Chromosome Segregation Does Not Depend on Previous Chromosome Associations During Early Meiosis

Each PMC analyzed at the MI stage was characterized by the presence of two barley univalents in double monosomic H. chilense-H. vulgare addition lines. Around 300 cells were observed in meiosis anaphase I. GISH analysis showed that both wild and cultivated barley univalents segregated simultaneously

FIGURE 3 | Detection of the subtelomeric HvT01 sequence in the wild and cultivated barley chromosomes in the 5Hch5Hv double monosomic addition line. DNA was counterstained with DAPI (blue). (a) GISH of both H. chilense (green) and H. vulgare (red) homoeologous chromosomes initiating pairing during early pachytene. (b) Detection of the subtelomeric HvT01 probe (green) on the same cell. (c) Merge image showing the subtelomeric HvT01 signal (overlay in white) on the terminal region of the unpaired homoeologous wild and cultivated barley chromosome arms (arrowed). Bar represents 10µm.

with wheat bivalents at stage anaphase I (**Figure 6**). All the different possible situations for chromosome segregation of the unpaired H. chilense and H. vulgare chromosomes were identified (**Figures 6a–e**, **Table 1**): (i) both barley chromosome were detected in both nuclei; (ii) only H. vulgare chromosome was detected in both nuclei; (iii) only H. chilense chromosome was detected in both nuclei; (iv) each barley chromosome was detected in each daughter nucleus; and (v) both barley chromosomes were detected in the same anaphase/telophase pole. These different situations for chromosome segregation of both barley chromosomes were found in all the genetic combinations (7Hch7Hv , 5Hch5Hv , and 7Hch5Hv double monosomic addition lines) in the wheat background, although the ratio between them varied depending on the genetic stock (**Table 1**). Nevertheless, no significant differences were found for H. chilense and H. vulgare chromosome segregation between the different genetic combinations (**Table 1**), despite the fact that the most frequent observation for the segregation of the wild and cultivated barley chromosomes was different in 7Hch7Hv addition line compared to 5Hch5Hv , and 7Hch5Hv addition lines (**Table 1**). Moreover, no significant differences were found on the behavior of the Hordeum chromosomes within the same F1 line. These results suggest that chromosome segregation in double H. chilense and H. vulgare monosomic addition lines in wheat occurred randomly, regardless the barley chromosomes added to the wheat background and whether or not chromosome associations took place previously in early meiosis.

Chromosomes delay was usually observed in double monosomic H. chilense and H. vulgare addition lines in late anaphase I/telophase I (**Figures 6f,g**), and the presence of chromatin across the equator during phragmoplast formation either from H. chilense, H. vulgare or both species was also observed (**Figures 6h–j**). Missegregation or chromosome breaks that occurred in anaphase I in the double monosomic H. chilense and H. vulgare addition lines cannot be distinguished from sister chromatids segregation unless using, among others, the HvT01 subtelomeric probe (**Figures 6k,l**).

Depending on chromosome segregation of both wild and cultivated barley univalents during meiosis I, the number of different genetic combinations in the PMC increased during MII, resulting in a wide range of meiotic phenotypes observed (nineteen different cases; **Table 2**). The most frequent H. chilense and H. vulgare chromosome combination observed for each genetic combination during telophase II was different depending on whether H. chilense and H. vulgare chromosomes were included in the same or in different homoeology group (**Figure 7**; **Table 2**), but no significant differences were found. Results suggested that both sister chromatids separation and misdivision did occur randomly independently of the barley chromosome combination.

## DISCUSSION

Little is known about how chromosomes recognize each other to correctly associate in pairs at early meiosis and recombine. This is a key question for plant breeders to transfer genetic variability from related species into a crop like wheat. The lack of recombination between cultivated wheat and alien chromosomes limits the transfer of novel traits from relatives to wheat because Ph1 suppresses homoeologous recombination between wheat and related species (Riley and Chapman, 1958; Sears, 1976). Different meiosis studies on chromosome pairing have been developed using wheat lines carrying an addition of one pair of homologous chromosomes or chromosome segments from one related species into wheat (Mikhailova et al., 1998; Maestra et al., 2002; Prieto et al., 2004a) or hybrids between wheat and relatives (Molnár-Láng et al., 2014; Rey et al., 2017). In this work, we have developed wheat lines carrying double monosomic chromosome additions for wild and cultivated barley for the same and for different homoeology group, respectively, which allowed us to track simultaneously by GISH a couple of extra homoeologous and non-homoeologous chromosomes from two different Hordeum species during early meiosis. These double monosomic addition lines can contribute to go deeper into the knowledge of how chromosomes recognize and associate in pairs

monosomic 7Hch7Hv addition line. (e) Double monosomic 7Hch5Hv addition line. (f) Both 7Hch5Hv barley chromosomes remained delayed. (g) Both 7Hch7Hv barley chromosomes remained delayed and a misdivision of chromosome 7Hv was also observed. One or both barley micronuclei were positioned in the equatorial region on telophase I in (h) Double monosomic 7Hch5Hv addition line, (i) Double monosomic 7Hch7Hv addition line, and (j) Double monosomic 7Hch7Hv addition line. The subtelomeric HvT01 probe was used in GISH experiments performed in cells in telophase I from the double monosomic 7Hch5Hv addition line to visualize (k) 5Hv chromosome misdivision or (l) 5Hv chromosome segregation. Bar represents 10µm.

in the wheat background in the presence of the Ph1 locus. In addition, most of the works carrying alien chromosomes in the wheat background are focused in meiosis metaphase I or later stages (Molnár-Láng et al., 2000; Silkova et al., 2014). The analysis of chromosome pairing focused only in metaphase I can result in an underestimation of homoeologous associations that might occur earlier in meiosis, as chromosomes might remain mostly as univalents due to the lack of homoeologous recombination. Few works analyzed the behavior of an extra pair of chromosomes at early stages of meiosis (Aragón-Alcaide et al., 1997; Prieto et al., 2004a; Valenzuela et al., 2012, 2013; Koo et al., 2016). Homologous barley chromosomes have been previously observed associated during early meiosis and metaphase I in disomic addition lines in wheat (Aragón-Alcaide et al., 1997; Calderón et al., 2014). In this study we have reported by GISH analysis that homoeologous wild and cultivated barley chromosomes can also fully associate in pairs in the wheat background during early meiosis and that such chromosome pairing occurred even in the presence of the Ph1 locus, although homoeologous barley chromosomes did not cross over and were always observed as univalent in metaphase I. The role of the Ph1 locus was recently narrowed down preventing recombination

between related chromosomes in interspecific hybrids (Moore, 2014; Martín et al., 2017). Our results clearly showed that the Ph1 locus does not hamper homoeologous chromosome associations but crossing over. Nevertheless, homoeologous recombination between related Aegilops geniculata and Ae. searsii has been detected in the wheat background in the presence of the Ph1 locus due to the presence of chromosome 5 Mg of Ae. geniculata, which harbors a homoeologous recombination promoter factor (Koo et al., 2016). Recombination frequencies between H. vulgare and H. bulbosum homoeologues have been previously detected but are lower than association frequencies (Zhang et al., 1999), probably because of non-chiasmate associations (Orellana, 1985). Homologous pairing has been also described in the absence of synapsis and meiotic recombination in Caenorhabditis elegans (Dernburg et al., 1998). Our results showed that homoeologous H. chilense and H. vulgare chromosomes associated in pairs in wheat in the absence of crossing over and that the Ph1 locus does not prevent such chromosome recognition and association between homoeologues. It is also worthy to mention that, although H. chilense and H. vulgare are phylogenetically quite distant, even included in two different sections among the Hordeum genus (Blattner, 2009), both species share a high degree

TABLE 1 | (A) Total number of PMCs scored at anaphase I showing the different combinations observed for both Hordeum chromosomes added to the wheat background. The most frequent observation per line and the total number of meiocytes examined are shown in bold. (B) Quantification of meiocytes (%) for each observation.

of similarities at the chromosomal level, as it has been reported between them and other species within this genus (Hernández et al., 2001; Aliyeva-Schnorr et al., 2016). Thus, other elements such as cohesins or the DNA sequence itself might play a major role on chromosome recognition and pairing at the onset of meiosis in a polyploidy like wheat.

So far, it is unclear whether initial recognition is mediated through protein-protein interactions, DNA base-pairing, or other chromosomal features. For example, a noncoding RNA (meiRNA-L) is responsible for the recombination-independent pairing of homologous loci in Schizosaccharomyces pombe (Ding et al., 2012). Other chromosomal features different from DNA/DNA recombinational interactions or RNA-mediated pairing have been proposed to be involved in the homologous recognition such as the pattern of cohesins distribution in the axial elements of unmatched meiotic chromosomes in mice and S. pombe (Ishiguro et al., 2011; Ding et al., 2016). Subtelomeres have been also reported as crucial to promote chromosome recognition and pairing between homologous chromosomes (González-García et al., 2006; Calderón et al., 2014). In our study, variability for the HvT01 subtelomeric sequence was found between H. chilense and H. vulgare chromosomes 5 and 7, particularly for the long arm of both chromosomes. We observed that although homoeologous chromosomes can potentially associate by the telomeres, subtelomeric DNA blocks might hamper homoeologous chromosome to correctly associate in pairs and thus, in the absence of homologs, chromosome recognition and association between homoeologues can occurred by the chromosome end where the subtelomeric repeats are shorter or absent. However, as meiosis progressed, the pairing signal initiated at these chromosome ends can be propagated along the whole chromosome, so that the homoeologues became fully associated by late pachytene. Thus, our results might suggest

that subtelomeres can play a key role in the specificity of chromosome recognition, restricting chromosome recognition to true homologs and therefore hampering homoeologous chromosomes to recognize each other and associate. The implication was that DNA sequence(s) within the subtelomeric region must be important for the process of initial homolog recognition and pairing, although further studies are required to reveal how subtelomeres take part in such important meiosis processes.

The peculiarities of univalent behavior in meiosis have been extensively studied in wheat aneuploids, particularly for the relation between the means of a chromosome segregation and its inclusion into a microspore (Sears, 1952; Marais and Marais, 1994; Friebe et al., 2005; Lukaszewski, 2010). The knowledge about univalent behavior in meiosis is necessary for the directed development of wheat lines carrying alien introgressions since univalent are subjected of incorrect division and segregation. Thus, abnormities in meiosis result in various modifications and/or in the loss of a transferred chromosome (Silkova et al., 2014). Univalents in meiosis have a tendency to misdivide (break) across their centromeres producing telocentric. This process has been deeply described in wheat (Sears, 1952; Steinitz-Sears, 1966; Friebe et al., 2005), and used to generate different cytogenetic stocks (Sears and Sears, 1978; Lukaszewski, 1993, 1997). The most common alien introgression in wheat, chromosome translocations, is the result of centric misdivision and fusion of misdivision products. Translocations between H. chilense and H. vulgare have been detected previously when the genomes of these species are in the same background (Prieto et al., 2001). Our results overview the univalent behavior of two homoeologous and non-homoeologous barley chromosomes in the wheat background. We observed that chromosome misdivisions and sister chromatids segregated randomly at anaphase I similarly to previous works (Friebe et al., 2005), and independently of whether or not related chromosomes associate in pairs in early meiosis.

In summary, homoeologous wild and cultivated barley chromosomes were observed fully associated in pairs in early meiosis in the presence of the Ph1 although crossing over did not occur at any time, as both chromosomes were always visualized as univalents during metaphase I. Whether or not homoeologous Hordeum chromosomes can crossover in the absence of the Ph1 locus remains to be elucidated. In addition, the role of the terminal chromosome regions in chromosome recognition and paring and the proteins interacting with these chromosomes ends will be key questions to shed light in future works.

#### HUMAN AND ANIMAL RIGHTS AND INFORMED CONSENT

This article does not contain any studies with human participants or animals.

#### REFERENCES

#### AUTHORS CONTRIBUTIONS

All authors contributed to this manuscript. MC and PP designed the research and performed the experiments. MC, MR, AM, and PP analyzed, discussed the results. PP and MC wrote the manuscript. All authors read and approved the manuscript.

#### ACKNOWLEDGMENTS