Single Cell Sequencing Dissects Meiotic Recombination And Haploid Induction In Maize

As a powerful technology,to date,single cell sequencing has been applied into many fields,such as embryogenesis,cancer,reproductive genetics,neurology,and immunology.However,it still faces a series of challenges in plant research nowadays.The cells or nuclei in gametophyte are the ideal material for single cell sequencing.In this study,the maize pollen at different stages was used to develop the plant single cell platform,to analyze the following projects: 1)Meiotic recombination plays an important role in completion of eukaryotic sexual reproduction and the generation of genome diversity.In order to study the mechanisms of homologous recombination in maize genome,single tetrad-stage microspores from F1 individuals were isolated and sequenced to study the recombination mechanism.2)Haploid induction(HI)is a natural process,which is a typical routine to induce maternal haploids with high efficiency in maize breeding.However,the underlying mechanism is still a mystery.By using single pollen nucleus sequencing,it was proposed that the chromosome fragmentation during pollen post meiosis could be a main factor for HI.Several important results in this study are summarized as following:1.A novel protocol was proposed to isolate single tetrad microspores for genome multiple displacement amplification(MDA).A total of 96 single microspores from 24 tetrads of F1 individuals were used for MDA and then the products were sequenced with 1.4X depth and 41% coverage of genome on average.A set of high-density SNPs were obtained.The median distance between adjacent SNPs was 235 bp.Through direct detection of genomic background exchanges,there were 924 fine-mapped crossovers(COs)totally identified across the whole genome.Averagely,38.5 COs occurs per one tetrad(meiosis).The recombination map was constructed in high-resolution to provide an insight of the uneven CO distribution.It was investigated that COs were more likely to happen near the gene regions,especially at the 5' and 3' ends of genes.Moreover,gene conversions(GCs)were uncovered in five CO tracts by Sanger sequencing,which implied that GCs always accompanied with CO tracts.A strong negative crossover interference and weak chromatid interference were observed in the microspore population.These findings provided an opportunity to understand the pattern of one-generation recombination and to improve the efficiency of maize breeding.2.A method was developed to isolate the single mature pollen nuclei in maize haploid inducer CAU5.A total of 66 single nuclei from 22 pollens and 72 microspores from18 tetrads were isolated for PCR-based whole-genome amplification(WGA).Through high-throughput sequencing and copy number variation(CNV)calling,a high frequency of pollen aneuploidy was observed at the three-nucleus stage(six of twenty two pollens)rather than at the tetrad stage(one of 72 microspores),which indicated that chromosome fragmentation initiates post meiosis in inducer's pollens.3.One approach was developed to isolate single sperm nuclei in the other inbreeding line.We isolated 50 and 52 single sperms from 25 B73 and 26 B73-inducer(80% B73 and 20% CAU2)pollens for PCR-based WGA,respectively.Through sequencing and CNV analysis,a high rate of aneuploidy(14/52 sperm)was observed in B73-inducer,while a distinct lower rate of aneuploidy(3/50 sperm)was detected in B73.The results implied that HI is related to pollen aneuploidy(chromosome fragmentation).4.To explain the relationship between pollen chromosome fragmentation and embryo HI,81 embryos and 81 endosperms of 88 maize 9-DAP(days after pollen)kernels were sequenced,which were from a cross between a regular maize inbred line and CAU5.Seven haploid embryos showed inducer-derived genome elimination,one embryo lost the most of inducer-derived genome,and three samples possessed small CNVs.Together,it was suggested that chromosome fragmentation initiating post pollen meiosis may cause HI.These results provided a novel insight into the mechanism of HI,and supplied much valuable information to breed elite haploid inducer.