Authors

Discussion

Leishmania exhibits extraordinary genomic plasticity through frequent gene and chromosome copy-number variation that underpins rapid adaptation, including the emergence of drug resistance. Understanding how these parasites duplicate their genome is therefore key to explaining how they balance plasticity with the genome stability required for survival. Using DNAscent to detect replication origins at single-molecule resolution and MFA-seq to map replication timing in bulk populations, we previously showed that origin usage is stochastic, and that replication timing is strongly compartmentalised both within and between chromosomes. However, it remains unclear whether replication initiates from a fixed set of loci or from a broader pool of potential sites used probabilistically in individual cells, and how reproducible origin firing time is from cell to cell. Here, we apply single cell whole genome sequencing followed by MFA-seq analysis (scMFA-seq) to map the replication timing in individual L. donovani promastigotes across S phase. scMFA-seq reveals that both intra-chromosomal and inter-chromosomal replication timing compartmentalisation are preserved at the single-cell level: replication initiation is largely confined to a single locus per chromosome overlapping the centromere, while larger chromosomes consistently replicate later than smaller ones. Quantifying cell-to-cell variability shows that replication timing follows a stochastic programme across the genome, yet cells initiate genome duplication in a coordinated manner around the expected S phase stage. Strikingly, analysing variability within individual cells indicates that ~50% of replication events deviate significantly from their predicted time, occurring either prematurely or with delay. Premature replication is positively associated with chromatin density, AT content, transcription initiation, and chromosome size, and negatively associated with GC content and late replication timing. Altogether, these data provide an unprecedented, single cell view of genome duplication programme in Leishmania, showing that a spatially constrained initiation landscape can still generate substantial temporal variability, an organisational logic that may help reconcile genome malleability with genome transmission.