Optimization And Application Research Of CENH3-Based Haploid Inducer Lines

Haploids,which contain only one set of genomes from either the father or the mother,are of great value in basic research and modern breeding.Obtaining a pure double haploid(DH)line can be achieved in just two generations after genome doubling,significantly shortening the breeding process.The production of haploids can be divided into two main methods: in vivo and in vitro.In vitro methods involve tissue culture regeneration of male and female gametophytes,while in vivo methods include heterologous pollen induction and haploid inducer lines.Haploid inducer lines are considered the most convenient and rapid technique for haploid production.One of the great potential haploid induction systems is the CENH3-based induction system.CENH3 is a histone H3 variant specific to the centromere in eukaryotes,playing a crucial role in centromere formation and function.Manipulating CENH3 function has been shown to create haploid inducer lines.The GFP-tailswap inducer line,where the N-terminal tail of CENH3 in Arabidopsis is replaced with the N-terminal tail of conventional H3.3 and GFP protein is fused to the modified CENH3 protein,can induce both paternal and maternal haploids with high flexibility.Since CENH3 function is highly conserved,this system is theoretically applicable to a wide range of other crops.However,the success of this method in other crops has not been as expected.Although CENH3-based haploid inducer lines have been successfully created in crops such as maize and wheat,they still have disadvantages such as low induction ability and low pollen viability,which hinder further application and research.Our study revealed that the pollen viability and haploid induction ability of the GFP-tailswap are highly sensitive to changes in ambient temperature,and the effect of temperature on both is independent.Based on these findings,we successfully designed strategies to optimize CENH3-based haploid inducer in both maternal and paternal parents through flexible manipulation of ambient temperature.We found that the fertility of the GFP-tailswap is highly sensitive to changes in ambient temperature.The normal physiological temperature range for Arabidopsis is 16 °C-25 °C,with the optimal growth temperature at 22 °C.Lowering the growth temperature of GFP-tailswap to 18 °C restored its fertility to over 80% of the wild type.Conversely,raising the temperature by 3 °C resulted in almost complete loss of fertility in GFP-tailswap.The infertility of the GFP-tailswap and its response to ambient temperature are primarily manifested in the development of female and male gametophytes,consistent with the reported role of CENH3 in meiotic chromosome segregation.Therefore,GFP-tailswap represents a temperature-sensitive male/female sterile line.The res1 mutant failed to restore the pollen viability and fertility of GFP-tailswap,indicating that the mechanism for the low-temperature recovery of pollen viability may differ from the reported mechanism of slowing down gametophyte development.In addition to fertility,we also discovered that GFP-tailswap is highly sensitive to ambient temperature,with its haploid induction capacity increasing from approximately 20% at 18 °C to around 80% at 25 °C.We further demonstrated that the sensitivity to temperature is a common feature of CENH3-based haploid inducer lines.We used the CRISPR technology to create a weak mutant,called cenh3-8,which showed no haploid induction ability under low-temperature and normal conditions but exhibited substantial haploid induction ability(13.6%)at 25 °C.Additionally,by manipulating the temperature before and after pollination,we found that the temperature effect on induction capacity is independent of its effect on gametophyte development.Based on our findings,we designed different strategies for manipulating temperature to optimize the induction of paternal and maternal haploids,focusing on simplifying the induction process and improving haploid efficiency.For the induction of paternal haploids,direct induction at 25 °C can be performed because nearly half of the female gametes still develop normally under this condition,eliminating the need for emasculation,and the induction efficiency is increased to over 77%.For the induction of maternal haploids,the strategy involves first recovering the activity of the induction line under low-temperature conditions and then pollinating the target plant with pollen from the recovered induction line.This approach resulted in a record-breaking haploid induction rate of maternal haploids of 24.8% or higher.Efficient haploid inducer lines have significant value in both basic research and seed industry applications.Using our optimized haploid induction strategies,we successfully created a versatile and permanent DH population that exhibits significant polymorphism in DNA sequence and epigenetic modifications(DNA methylation).The DH lines show pronounced phenotypic segregation in various aspects,including seed size,flowering time,and dormancy.Therefore,this population can be used to mapping conventional quantitative trait loci(QTL)and epigenetic QTL,as well as important questions regarding the interaction between genetic loci and epigenetic loci.In summary,we have identified ambient temperature as a key factor influencing CENH3-based haploid induction systems.By flexibly manipulating ambient temperature,we can optimize the production of maternal and paternal haploids in terms of haploid induction difficulty and efficiency.Given the conservation of CENH3 function across species,our research provides new clues for the application of this system in a wider range of crops.