Using Single Cell ATAC-seq Method To Analyze Recombination In Maize Pollen Nuclei

Meiosis is the process for eukaryotes to produce haploid gamete cells.Crossover between homologous chromosomes takes place in the meiotic prophase ? stage and results in genetic recombination,which is the basis of normal separation between homologous chromosomes and the crucial source of genetic variation.Current research on crossover distribution in plants mainly relies on cellular immunostaining or genotyping by sequencing from hybrid progeny populations,however,which is laborious and expensive,and not suitable for high-throughput study.With the development of sequencing technology,single cell genomic sequencing provides an efficient approach for genetic variation research.Single-cell ATAC-seq is often used to study the chromatin accessibility with the library from transposase-accessible chromatin,with the advantages of easy operation and low cost.Thus,we applied single-cell ATAC-seq to construct genomic libraries and detected the distribution of recombination in maize pollen.In this study,we isolated the nuclei from maize F1 pollen from B73 x Mo17 hybrid using the gradient centrifugation method.Combining flow cytometry and single-cell ATAC-seq method,we constructed 191 single-nucleus libraries.The mitochondrial and chloroplast genome contamination rate was 0.01%.The average sequencing depth of the nucleus was 0.01 × and the doublet ratio was 6.8%.The length distribution of the inserted fragments showed a periodic distribution rule,in which peak distance was roughly 200 bp.Simultaneously,we extracted a total of 6 752 569 SNPs from the parental B73 and Mo17 genomes using the maize hapmap3 data.34 nuclei showed which read coverage of SNP sites greater than 1 000.Each nucleus had an average of 2 047 SNPs.However,the ratio of SNPs genotype showed a deviation from the normal 1:1 ratio.The average crossover count of the nuclei was 30.47.Finally,we got 12 crossover hotspots and 6 crossover coldspots.Thus,we used single cell ATAC-seq method to construct pollen nuclei genomic libraries and performed sequencing.And due to the low library genomic coverage,we were unable to perform fine-scale recombination study.Moreover,we also used an Arabidopsis transient method to transform the 35 S promoter-driven vector into Arabidopsis seedlings.GUS staining and fluorescence microscopy analysis showed that the transformed genes were mainly expressed in cotyledon mesophyll cells,which was an implication for subsequent plant singlecell CRISPR screening.