[en] Genetic recombination is an important feature of begomoviruses, single stranded DNA virus belonging to family Geminiviridae. Extremely complex and widely distributed recombinations have been observed upon begomovirus infections [1]. Apart from virusvirus recombination, a recent study has revealed spontaneous formation of hybrid DNA minicircles composed of begomovirus and plant genomic DNA sequences [2]. We have recently developed CIDER-Seq (Circular DNA Enrichment sequencing), a pipeline for the unbiased enrichment and highly accurate long-read sequencing of extra-chromosomal circular DNA (eccDNA) molecules [3]. Here we demonstrate the utilization of CIDER-Seq to sequence and characterize recombinant plant-geminivirus eccDNAs. Arabidopsis plants, agroinoculated with cabbage leaf curl virus (CaLCuV), were subjected to modified rolling circle amplification-mediated enrichment of eccDNAs. SMRT (Single Molecule Real Time) libraries were then prepared and sequenced on PacBio Sequel platform. BLAST analysis indicated that almost 80% of the reads contained CaLCuV sequences. Analysis of these reads lead to the identification of 74 Arabidopsis-CaLCuV recombinants, among which 22% (16/74) belonged to CaLCuV DNA-A and 78% (58/74) belonged to CaLCuV DNA-B. The size of recombinants ranged from 624nt to 7,535nt, with an average recombinant size of 3,520nt. Overall 82% (61/74) of recombinant fragments contained chromosomal DNA, while 15% (11/74) and 3% (2/74) had chloroplast and mitochondrial DNA origins, respectively. These host sequences from recombinant fragments did not share significant similarities or patterns, except all being AT rich (~70%) and almost all lacking open reading frames (ORFs). However, in two recombinants we were able to identify complete ORFs coding for Sucrase/ferredoxin-like family protein and DNA double-strand break repair protein. Further experiments are required to characterize the frequency and the role of host-geminivirus recombination events during geminivirus infection.
