Periodic Reporting for period 1 - Crossover control (New insights into wheat meiosis: Crossover resolution in the absence of the Ph1 locus)
Periodo di rendicontazione: 2016-03-01 al 2018-02-28
This project aimed to increase the crossover frequency in bread wheat, which would result in a higher number of recombination events and would be a powerful tool for plant breeding. In the next 50 years, we will require as much wheat as has been produced since the beginning of agriculture. There are 1.5 billion dependent poor in the world, of which 300 million living in countries surrounding Europe are at risk of starvation in the future. Finding solutions to address the problems of wheat production will be a dominant topic in European plant science. Future breeding strategies for wheat will require exploitation of diversity in wild relatives, and manipulation of the Ph1 locus will be a powerful tool to facilitate introgression of traits from these related species. The aim of this proposal of to exploit the Ph1 locus in wheat as a tool to understand CO formation with the final goal of increasing recombination between the chromosomes of wheat and its wild relatives. The importance of wheat as a crop, and the need to exploit its wild relatives as donors for economically important traits – yield, disease resistance, increased drought tolerance - in wheat breeding programmes, is the main drive to uncover the mechanism of Ph1, a key influence in genetic crossing, and regulate its activity.
Objective 1: Evaluation of the effect of environmental factors in CO frequency in wheat and wild relative species: To assess the effect of nutrient concentration on homologous and homoeologous CO frequency in meiotic metaphase I, we added a modified Hoagland solution to the soil in which wheat and wheat-rye hybrids. In the absence of Ph1, we can increase the CO frequency by altering environmental factors: we show that higher nutrient levels in the soil or lower temperatures increase the level of both homologue and homoeologue COs. In the presence of Ph1, no significant increase in CO number was observed.
Objective 2: Exploitation of the ZIP4 homologue within the wheat Ph1 locus: The wheat Ph1 locus promotes accurate synapsis and CO of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, COs do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, COs do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.
Objective 3: Identification of the macronutrient responsible for the increase in the CO number observed 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 CO between wheat and wild relative chromosomes. Hybrids between wheat carrying a 70Mb deletion (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 and up to a mean of 12 COs per cell by treating the plants with Hoagland solution. More recently, it was shown that the major meiotic CO gene ZIP4 (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). 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. 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 Mg2+ supplementation in improving the success of genetic material introgression from wild relatives into wheat.
Objective 2: Exploitation of the ZIP4 homologue within the wheat Ph1 locus: The wheat Ph1 locus promotes accurate synapsis and CO of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, COs do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, COs do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.
Objective 3: Identification of the macronutrient responsible for the increase in the CO number observed 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 CO between wheat and wild relative chromosomes. Hybrids between wheat carrying a 70Mb deletion (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 and up to a mean of 12 COs per cell by treating the plants with Hoagland solution. More recently, it was shown that the major meiotic CO gene ZIP4 (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). 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. 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 Mg2+ supplementation in improving the success of genetic material introgression from wild relatives into wheat.
Optimisation of a chromosomal spread technique using wheat and barley root tips: Chromosomal spread techniques allow the identification and characterisation of chromosomes of different species. Both colchicine and ice-cold water have been widely used in chromosomal spread techniques. However, in my experience, the success of this technique using these conditions is quite low, and the method is time-consuming, resulting in small amounts and bad quality of chromosomal spreads. Considering this, I went to the Ian King’s group (University of Nottingham, UK) to learn how to use the nitrous oxide gas to obtain fast and good quality chromosomal spreads in wheat root tips. After learning this technique, I optimized it at the John Innes Centre making this technique available for everyone. Moreover, I carried out the identification and comparison of individual chromosomes of three Hordeum chilense accessions, Hordeum vulgare and Triticum aestivum by fluorescence in situ hybridisation.
Collaboration in the development of a speed breeding protocol used to accelerate crop research and breeding, evaluating the effect that speed breeding might have on wheat chromosome pairing at metaphase I in meiosis in the presence and absence of the Ph1 locus. Wheat lacking Ph1 and wheat-rye hybrids carrying and lacking Ph1 were grown in speed breeding and control conditions, and observed no significant differences in chromosome pairing and recombination in meiocytes at metaphase I. The chromosome behaviour suggests that both wheat and wheat-rye hybrids are cytologically stable under speed breeding conditions.
Collaboration in the development of a speed breeding protocol used to accelerate crop research and breeding, evaluating the effect that speed breeding might have on wheat chromosome pairing at metaphase I in meiosis in the presence and absence of the Ph1 locus. Wheat lacking Ph1 and wheat-rye hybrids carrying and lacking Ph1 were grown in speed breeding and control conditions, and observed no significant differences in chromosome pairing and recombination in meiocytes at metaphase I. The chromosome behaviour suggests that both wheat and wheat-rye hybrids are cytologically stable under speed breeding conditions.