A separation-of-function ZIP4 wheat mutant allows crossover between related chromosomes and is meiotically stable

Many species, including most flowering plants, are polyploid, possessing multiple genomes. During polyploidisation, fertility is preserved via the evolution of mechanisms to control the behaviour of these multiple genomes during meiosis. On the polyploidisation of wheat, the major meiotic gene ZIP4 duplicated and diverged, with the resulting new gene TaZIP4-B2 being inserted into chromosome 5B. Previous studies showed that this TaZIP4-B2 promotes pairing and synapsis between wheat homologous chromosomes, whilst suppressing crossover between related (homoeologous) chromosomes. Moreover, in wheat, the presence of TaZIP4-B2 preserves up to 50% of grain number. The present study exploits a ‘separation-of-function’ wheat Tazip4-B2 mutant named zip4-ph1d, in which the Tazip4-B2 copy still promotes correct pairing and synapsis between homologues (resulting in the same pollen profile and fertility normally found in wild type wheat), but which also allows crossover between the related chromosomes in wheat haploids of this mutant. This suggests an improved utility for the new zip4-ph1d mutant line during wheat breeding, compared to the previously described CRISPR Tazip4-B2 and ph1 mutant lines. The results also reveal that loss of suppression of homoeologous crossover between wheat chromosomes does not in itself reduce wheat fertility when promotion of homologous pairing and synapsis by TaZIP4-B2 is preserved.

A separation-of-function ZIP4 wheat mutant allows crossover between related chromosomes and is meiotically stable

ABSTRACTMany species, including most flowering plants, are polyploid, possessing multiple genomes. During polyploidisation, fertility is preserved via the evolution of mechanisms to control the behaviour of these multiple genomes during meiosis. On the polyploidisation of wheat, the major meiotic gene ZIP4 duplicated and diverged, with the resulting new gene TaZIP4-B2 being inserted into chromosome 5B. Previous studies showed that this TaZIP4-B2 promotes pairing and synapsis between wheat homologous chromosomes, whilst suppressing crossover between related (homoeologous) chromosomes. Moreover, in wheat, the presence of TaZIP4-B2 preserves up to 50% of grain number. The present study exploits a ‘separation-of-function’ wheat Tazip4-B2 mutant named zip4-ph1d, in which the Tazip4-B2 copy still promotes correct pairing and synapsis between homologues (resulting in the same pollen profile and fertility normally found in wild type wheat), but which also allows crossover between the related chromosomes in wheat haploids of this mutant. This suggests an improved utility for the new zip4-ph1d mutant line during wheat breeding exploitation, compared to the previously described CRISPR Tazip4-B2 and ph1 mutant lines. The results also reveal that loss of suppression of homoeologous crossover between wheat chromosomes does not in itself reduce wheat fertility when promotion of homologous pairing and synapsis by TaZIP4-B2 is preserved.

A Duplicated Copy of the Meiotic Gene ZIP4 Preserves up to 50% Pollen Viability and Grain Number in Polyploid Wheat

Although most flowering plants are polyploid, little is known of how the meiotic process evolves after polyploidisation to stabilise and preserve fertility. On wheat polyploidisation, the major meiotic gene ZIP4 on chromosome 3B duplicated onto 5B and diverged (TaZIP4-B2). TaZIP4-B2 was recently shown to promote homologous pairing, synapsis and crossover, and suppress homoeologous crossover. We therefore suspected that these meiotic stabilising effects could be important for preserving wheat fertility. A CRISPR Tazip4-B2 mutant was exploited to assess the contribution of the 5B duplicated ZIP4 copy in maintaining pollen viability and grain setting. Analysis demonstrated abnormalities in 56% of meiocytes in the Tazip4-B2 mutant, with micronuclei in 50% of tetrads, reduced size in 48% of pollen grains and a near 50% reduction in grain number. Further studies showed that most of the reduced grain number occurred when Tazip4-B2 mutant plants were pollinated with the less viable Tazip4-B2 mutant pollen rather than with wild type pollen, suggesting that the stabilising effect of TaZIP4-B2 on meiosis has a greater consequence in subsequent male, rather than female gametogenesis. These studies reveal the extraordinary value of the wheat chromosome 5B TaZIP4-B2 duplication to agriculture and human nutrition. Future studies should further investigate the role of TaZIP4-B2 on female fertility and assess whether different TaZIP4-B2 alleles exhibit variable effects on meiotic stabilisation and/or resistance to temperature change.

A duplicated copy of the meiotic gene ZIP4 preserves up to 50% pollen viability and grain number in polyploid wheat

SummaryAlthough most flowering plants are polyploid, little is known of how the meiotic process evolved to stabilise and preserve polyploid fertility. On wheat polyploidisation, the major meiotic gene ZIP4 on chromosome 3B duplicated onto 5B and subsequently diverged. This 5B meiotic gene copy (TaZIP4-B2) was recently shown to promote homologous pairing, synapsis and crossover, and suppress homoeologous crossover. We therefore suspected that these stabilising effects on meiosis could be important for the preservation of wheat polyploid fertility.A CRISPR Tazip4-B2 mutant was exploited to assess the contribution of the 5B duplicated ZIP4 copy in maintaining pollen viability and grain setting.Analysis demonstrated abnormalities in 56% of meiocytes in the Tazip4-B2 mutant, with micronuclei in 50% of tetrads, reduced size in 48% of pollen grains and a near 50% reduction in grain number. Further studies showed that most of the reduced grain number resulted from pollination with less viable pollen, suggesting that the stabilising effect of TaZIP4-B2 on meiosis has a greater consequence in subsequent male, rather than female gametogenesis.These studies reveal the extraordinary value of the wheat chromosome 5B TaZIP4-B2 duplication to agriculture and human nutrition. Future studies should assess whether different TaZIP4-B2 alleles exhibit variable effects on meiotic stabilisation and/or resistance to temperature change.

Quantitative Trait Loci Mapping for Spot Blotch Resistance in Two Biparental Mapping Populations of Bread Wheat

Spot blotch (SB), caused by Bipolaris sorokiniana, is a major fungal disease of wheat in South Asia and South America. Two biparental mapping populations with 232 F2:7 progenies each were generated, with CIMMYT breeding lines CASCABEL and KATH as resistant parents and CIANO T79 as the common susceptible parent. The two populations were evaluated for field SB resistance in CIMMYT’s Agua Fria station for three consecutive cropping seasons, with artificial inoculation. Genotyping was done with the DArTseq platform and approximately 1,500 high quality and nonredundant markers were used for quantitative trait loci (QTL) mapping. In both populations, a major QTL was found on chromosome 5A in the Vrn-A1 region, explaining phenotypic variations of 13.5 to 25.9%, which turned up to be less- or nonsignificant when days to heading and plant height were used as covariates in the analysis, implying a disease escape mechanism. Another major QTL was located on chromosome 5B in CASCABEL, accounting for 8.9 to 21.4% of phenotypic variation. Minor QTL were found on 4A and 4B in CASCABEL; 1B, 4B, and 4D in KATH; and 1B, 2B, and 4B in CIANO T79. Through an analysis of QTL projection onto the IWGSC Chinese Spring reference genome, the 5B QTL in CASCABEL was mapped in the Sb2 region, delimited by the single nucleotide polymorphism marker wsnp_Ku_c50354_55979952 and the simple sequence repeat marker gwm213, with a physical distance of about 14 Mb to the Tsn1 locus.

Molecular cytological analysis of alien introgressions in common wheat lines derived from the cross of TRITICUM AESTIVUM with T. kiharae

Background Triticum kiharae (AtAtGGDD, 2n = 42) is of interest for the improvement of bread wheat as a source of high grain protein and gluten content, as well as resistance to many diseases. The use of T. kiharae for the improvement of T. aestivum L. is complicated by the fact that the homology degree of their genomes is low and this leads to an unbalanced set of chromosomes in the gametes of its first generations and the elimination of some genotypes. The aim of this study was to analyze the nature of alien introgressions and their effect on the cytological stability of hybrids obtained from crossing of bread wheat varieties with T. kiharae. Results Using C-banding, the presence of entire chromosomes of T. kiharae in the karyotypes of hybrid lines (intergenomic substitution 2G/2B), chromosome arms (centric translocation Т2AtS:2AL) and large inserts in the form of terminal translocations involving chromosomes of 1st, 3rd and 5th homoeologous groups of B- and G-genomes were found. Molecular markers revealed short introgression of T. kiharae into the genome of common wheat varieties. The highest introgression frequency was shown for 1A, 1B, 2A, 5B, and 6A chromosomes, while no foreign chromatin was detected in 4A and 4B chromosomes. A high level of cytological stability (a meiotic index of 88.18–93.0%) was noted for the majority of introgression lines. An exception was found for the lines containing the structural reorganization of chromosome 5B, affecting the main genes of chromosome synapsis in terms of their functioning. Conclusions During the stabilization of hybrid karyotypes, the introgression of genetic material from T. kiharae into the genome of T. aestivum occurs in the form of short fragments detectable only by molecular markers and in the form of whole chromosomes (intergenomic substitution) and their large fragments (centric and terminal translocations). The level of cytological stability achieved in F10 by the majority of introgression lines ensures the formation of functional gametes sufficient for the successful reproduction of the obtained hybrids.

Homoeologous Recombination: A Novel and Efficient System for Broadening the Genetic Variability in Wheat

Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination. The presence of the Pairing homoeologous 1 (Ph1) gene in the long arm of wheat chromosome 5B allows only homologous chromosomes to pair and recombine, resulting in diploid inheritance of polyploid wheat. Previously, we identified a potent homoeologous pairing promotor gene(s) (Hpp-5Mg); its carrier chromosome 5Mg derived from Aegilops geniculata and its wheat homoeologous chromosome 5D freely recombined in the presence of the Ph1 gene. In this study, we investigated the effect of Hpp-5Mg on homoeologous recombination in the absence of Ph1. In Hpp-5Mg/ph1bph1b plants, we observed a vast genome-wide increase in homoeologous recombination and multiple crossovers (CO), including CO breakpoints in proximal regions of the chromosomes where recombination is known to be suppressed. We tested the efficacy of Hpp-5Mg/ph1bph1b-induced homoeologous recombination by producing new recombinants for the wheat streak mosaic virus resistance gene, Wsm3, present in the wheat-Thinopyrum intermedium Robertsonian translocation (RobT T7BS.7S#3L). A recombination frequency of 6.5% was detected by screening the progenies double monosomic for T7BS.7S#3L and 7B by genomic in situ hybridization. This recombination frequency was about 100-fold higher compared with the recombinant frequency of 0.06% observed by using ph1b-induced homoeologous recombination alone. Our results indicate that chromosome 5Mg promotes homoeologous recombination between wheat and wild wheat relative chromosomes, which helps in the generation of pre-breeding materials thereby accelerating wheat crop improvement.

Genome-Wide Association Studies for Spot Blotch (Cochliobolus sativus) Resistance in Bread Wheat Using Genotyping-by-Sequencing

Spot blotch is a severe biotic menace of wheat caused by Cochliobolus sativus (syn. Bipolaris sorokiniana). Spot blotch is liable to major yield losses in warm humid regions. A genome-wide association study using genotyping-by-sequencing (GBS) markers was conducted to identify genomic regions associated with spot blotch resistance in a diversity panel of 159 spring wheat genotypes. In total, 87,096 GBS markers covering the whole genome, with an average polymorphism information content value of 0.276, were applied. Linkage disequilibrium (LD) analysis indicated that the LD decay extent was approximately 100 Mbp. The panel was evaluated for disease severity (DS) and area under disease progress curve (AUDPC) for 2 years. In total, 24 marker-trait associations (MTA) were identified for DS and AUDPC of spot blotch, with 11 on chromosome 5B, 3 on 3A, 2 on 6B, and 1 each on 1A, 2A, 1D, 2D, 4B, 5A, 7A, and 7B. A marker on chromosome 7B significantly explained 14% of the phenotypic variation of spot blotch severity as well as 11% of AUDPC. Five markers—three on chromosome 5B, one on 3A, and one on 7B—were associated with both DS and AUDPC with R2 ranging from 8 to 12%. Significant MTA can be utilized to develop wheat germplasm with resistance to spot blotch.

Magnesium increases homoeologous crossover frequency in ZIP4 (Ph1) mutant wheat-wild relative hybrids

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 70Mb 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 70Mb 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 analysed. 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 analysed hybrids, when plants were irrigated with a 1mM Mg2+ solution. These observations suggest a role for magnesium supplementation in improving the success of genetic material introgression from wild relatives into wheat.