ZIP4 is required for normal progression of synapsis and for over 95% of crossovers in wheat meiosis

Abstract

Tetraploid (AABB) and hexaploid (AABBDD) wheat have multiple sets of similar chromosomes, with successful meiosis and preservation of fertility relying on synapsis and crossover (CO) formation only taking place between homologous chromosomes. In hexaploid wheat, the major meiotic gene TaZIP4-B2 (Ph1) on chromosome 5B, promotes CO formation between homologous chromosomes, whilst suppressing COs between homeologous (related) chromosomes. In other species, ZIP4 mutations eliminate approximately 85% of COs, consistent with loss of the class I CO pathway. Tetraploid wheat has three ZIP4 copies: TtZIP4-A1 on chromosome 3A, TtZIP4-B1 on 3B and TtZIP4-B2 on 5B. Here, we have developed single, double and triple zip4 TILLING mutants and a CRISPR Ttzip4-B2 mutant, to determine the effect of ZIP4 genes on synapsis and CO formation in the tetraploid wheat cultivar 'Kronos'. We show that disruption of two ZIP4 gene copies in Ttzip4-A1B1 double mutants, results in a 76-78% reduction in COs when compared to wild-type plants. Moreover, when all three copies are disrupted in Ttzip4-A1B1B2 triple mutants, COs are reduced by over 95%, suggesting that the TtZIP4-B2 copy may also affect class II COs. If this is the case, the class I and class II CO pathways may be interlinked in wheat. When ZIP4 duplicated and diverged from chromosome 3B on wheat polyploidization, the new 5B copy, TaZIP4-B2, could have acquired an additional function to stabilize both CO pathways. In tetraploid plants deficient in all three ZIP4 copies, synapsis is delayed and does not complete, consistent with our previous studies in hexaploid wheat, when a similar delay in synapsis was observed in a 59.3 Mb deletion mutant, ph1b, encompassing the TaZIP4-B2 gene on chromosome 5B. These findings confirm the requirement of ZIP4-B2 for efficient synapsis, and suggest that TtZIP4 genes have a stronger effect on synapsis than previously described in Arabidopsis and rice. Thus, ZIP4-B2 in wheat accounts for the two major phenotypes reported for Ph1, promotion of homologous synapsis and suppression of homeologous COs.

Keywords: Ph1; Triticum turgidum; ZIP4; crossover; meiosis; synapsis; tetraploid wheat.

Figure 1

Representative images of chromosomes at metaphase I of meiosis from meiocytes of Kronos plants with differing TtZIP4 genotypes (A) Wild-type Kronos; (B) Kr3161 TtZIP4-A1B1B2; (C) Ttzip4-A1 single mutant; (D) Ttzip4-B1 single mutant; (E) Ttzip4-B2 single mutant (ph1c); (F) CRISPR Ttzip4-B2 single mutant; (G) Ttzip4-A1B1 double mutant; (H) Ttzip4-A1B2 double mutant; (I) Ttzip4-B1B2 double mutant; (J) Ttzip4-A1B1B2 triple mutant. Examples of univalent chromosomes (univ.), rod bivalents (rod), ring bivalents (ring), single chiasmata (X) and double chiasmata (XX) are indicated with arrows. Note the greatly increased univalence in the Ttzip4-A1B1 double mutant (G) and complete univalence in the Ttzip4-A1B1B2 triple mutant (J). Scale bar, 10 μm.

Figure 2

Column chart showing genotypic effects on meiotic metaphase I chromosomes of tetraploid wheat Ttzip4 mutants compared with Kr3161 wild type control plants (ZIP4-A1B1B2). Numbers of univalents, rod and ring bivalents and single chiasmata are shown. Numbers of multivalents and double chiasmata are not shown. Note greatly increased numbers of univalents (~18 out of 28 chromosomes) and low chiasma frequency in zip4-A1B1 double mutants, and virtually complete univalence and almost no chiasmata in the zip4-A1B1B2 triple mutants.

Figure 3

Immunolocalization of the meiotic proteins ASY1 (magenta) and ZYP1 (green) combined with the telomeres (red) labeled by FISH in tetraploid wheat with different copies of TtZIP4 (TtZIP4-A1B1B2 (wild-type control), zip4A1B1 (double mutant) and zip4-A1B1B2 (triple mutant). DNA DAPI staining in blue. Scale bar represents 10 μm. (A) Synapsis during the early telomere bouquet stage. Initiation of synapsis is observed at the telomere bouquet in the wild type and double mutant showing the typical ZYP1 polarization. However, in the triple mutant, in the absence of all ZIP4 copies, synapsis initiation is mostly delayed and only short stretches of ZYP1 are observed dispersed throughout the nucleus. (B) Progression of synapsis after bouquet dispersal. Synapsis in the control and double mutant is almost completed at this stage, while synapsis is far from completion in the absence of all ZIP4 copies, as illustrated by the large amount of ASY1 labeling still visible.