Rhynchospora_tenuis_project

Cutstom scripts for the manuscript of R. tenuis project.

Zhang, Meng, et al. "Sex without crossovers mimics clonal reproduction in the holocentric plant Rhynchospora tenuis." bioRxiv (2026): 2026-01. doi: https://doi.org/10.64898/2026.01.17.700054

Synteny analyses

syri_plot.sh: DNA collinearity by SyRI and plotsr. Reference sctipts for Figure 1b and Extended Figure 2.

run_genespace.R: Genome syteny based on orthologous genes. Reference script for running GeneSpace in Figure 1a.

Divergence time estimation of two haplotypes

divergence_time_estimation/Fig_1f_estimate_divergence_time.R: Divergence time calculation and plot for Figure 1f.

• The input Ks values between two haplotypes was computed by CoGe based on the CDS sequences annotated by Helixer. All Ks of the CDSs overlapped with TE annotations were removed. Refer to the script divergence_time_estimation/remove_TE_CDS.sh
• The SNP input was derived from SyRI from above synteny analysis, then the SNP density (SNP per Mb) were calculated as the input.
• The LTR divergence used the output from SubPhaser. Please check divergence_time_estimation/extract_LTR_insertion_time_from_SubPhaser.sh

Single cell analyses of pollen nuclei

The genotyping of single pollen nuclei used the same pipeline as described in Castellani, M., Zhang, M. et al. The plots in Figure 3a was also part of this pipeline (hapCO_identification.R in the pipeline GitHub page).

segregation_bias_ChiSquareTest.R: Non-mendelian segregation validation in Figure 3d.

Single cell ATAC-seq analyses of embryo and endosperm nuclei

The pipeline is similar to single cell pipeline for pollen nuclei in general with minor modifications. The complete pipeline is available in the folder embryo_scATAC_analyses/. This pipeline took the 10X scATAC-seq of embryo nuclei from R. tenuis REC accession as an example. The endosperm analyses has no difference to it.

plot_AF_along_chrs.R: Figure 4j.

plot_AF_along_chrs.R: Figure 4k.

F1 genotyping

The genotyping of selfed F1 plants used the same pipeline as described in Castellani, M., Zhang, M. et al. We uploaded an example run here with script name as tiger_pipeline.sh. Please note that the key input of this pipeline is ´VCF´ file that contains the SNPs of F1 WGS reads aligned to one of the haplotypes of R. tenuis (REC). Input markers are genotyping markers defined by haplotype-specific SNPs, which was called from the alignments of deep-sequenced R. tenuis (REC) short-reads to haplotype 1 assembly.

plottings/Fig_3b_plot_F1_AF.R: Script of Figure 3b for allele frequency along chromosomes and its density margin plots of a F1 individual.

ChIP-seq pipeline and relevant analyses

ChIP_and_methylation_metaplots/ includes the pipeline for DNA methylation data analyses using Bismark and other following plotting scripts. The other ChIP-seq data and analyses were available in our previous publication Hofstatter et al..

2_metaplot.sh: Scripts for the metaplots in Extended Data Fig. 1c,d.

4_correlation.R Correlation analysis of epigentic signals in the metaplots (Extended Data Fig. 1c,d) - CENH3, H3K4me3, H3K9me2, CG, CHG, CHH between R. pubera, R. breviuscula, R. tenuis (REC) in Supplementary Table 2.

Phylogenetic relation of R. tenuis accessions

phylogeny_based_on_SCOs/ includes the scripts to construct the phylogenetic tree in Extended Data Fig. 4b based on single copy orthologs (SCOs) among R.breviuscula, R. austrobrasiliensis, and all haplotypes of R. tenuis. SCOs were extracted from the output of OrthoFinder in GENESPACE (check the above section # Synteny analyses).

Pseudogene analyses

search_pseudogenes/ includes the complete pileline of pseudogene analyses with Helixer gene annotations and RNA alignments as inputs. RNA alignments were done by mapping RNA short-read sequencing data to diploid R. breviuscula and R. tenuis (REC) assembly using STAR. Please note that the results can differ a lot when including the CDSs of TEs. So it is highly recommended to remove the coding sequences of TEs when searching pseudogenes, which are usually largely accumulated in many plant genomes.

03_report_pseudogenes.R: Scripts for the plots in Extended Data Fig. 5c.

Gene conversion detection based on F1 WGS data

detect_gene_conversion.R: The script shows how we detect gene conversion events. Input file is from our F1 genotyping pipeline with TIGER. The input file is input_corrected.txt from the first step of tiger_pipeline.sh in #F1 genotyping, which contains six fields:

1 chrom  2 pos  3 hap1_allele  4 hap1_count  5 hap2_allele  6 hap2_count

Although the detection pipeline output some potential gene conversions, they are not reliable after careful manual check in IGV, because we never observed a single read that can support gene conversion events. The mean coverage of our F1 samples is 1x-3x. So our result does not exclude the possibility of gene conversion events in R. tenuis, considering the sequencing limitation of our data.

Other

plottings/ExDataFig5_plot_haplotype_genome_difference.R: Extended Data Fig. 5a,b

plottings/SuppFig14_plot_austro_aln_to_tenuis_at_fusion_position.py Supplementary Fig. 14b, d. The input file was a few fields from PAF file that from minimap2 output by mapping R. austrobrasiliensis chromosomes/scaffolds to R. tenuis chromosome 1. Please read the script to see what information is needed.

Final comment

Please open an issue if you have any doubts about the scripts and data processing questions regarding our publication. But before doing it, please make sure you cannot find a solution in the Methods section in our publication.