Fig 1.
The mobilome-seq approach in plants.
A schematic view of the main steps involved in the selection and amplification of the extrachromosomal circular molecules in plants. After DNA extraction, linear DNA molecules are digested and circular molecules are randomly amplified using rolling circle amplification. This DNA material is used for high-throughput sequencing. Mobilome-seq data analysis consists in characterizing the depth of coverage (DOC) of mapped reads and the presence of split reads (SRs) at TE loci and the detection of de novo assembled scaffolds corresponding to these TEs.
Fig 2.
Mobilome-seq detection of EVD, a known active retrotransposon in Arabidopsis.
(A) Southern blot experiment using HindIII-digested eccDNAs amplified from A. thaliana WT and epi12 flowers and siliques and detected with a probe specific for the EVD retrotransposon active in the line epi12. RCA: rolling circle amplification. The ethidium bromide (EtBr) gel picture is shown as a loading control. (B) Abundance of reads mapping at TE-annotated loci in the A. thaliana epi12 line mobilome-seq library. Each dot represents the normalized coverage per million mapped reads per all TE-containing 100bp windows obtained after aligning the sequenced reads on the A. thaliana reference genome. Blue dots indicate the windows corresponding to annotated EVD genomic loci. (C) Detail of the depth of coverage of total mapped reads (DOC) and split reads (SR) abundance of the A. thaliana epi12 and WT mobilome-seq data at the EVD locus on chromosome 5 (blue bar). Grey peaks: read abundance (not normalized), DOC: depth of coverage for all aligned reads, SRs: split reads, WT: wild type siliques, epi12: epi12 siliques. Maximum coverage is indicated on the right. Colors indicate the presence of SNPs.
Fig 3.
Mobilome-seq detection of Tos17, a known active retrotransposon in rice callus.
(A) Abundance of reads mapping at TE-annotated loci in the O. sativa WT callus mobilome-seq library. Each dot represents the normalized coverage per million mapped reads per all TE-containing 100bp windows obtained after aligning the sequenced reads on the O. sativa reference genome. Green dots indicate the windows corresponding to annotated Tos17 genomic loci. (B) Detail of the depth of coverage of total mapped reads (DOC) and split reads (SRs) abundance of the O. sativa WT callus and leaf mobilome-seq library at the Tos17 locus on chromosome 7 (green bar). Legend as in Fig 2C. (C) Detection of circular forms of Tos17 using inverse PCR with primers localization depicted on the right (black bar: Tos17 element, arrows: PCR primers, grey boxes: LTRs). Upper gel: PCR amplification of Tos17 circles, middle gel: control PCR for Tos17 detection, lower gel: PCR using eEF1α primers as loading control. (D) Dotter alignment of the scaffold #29 obtained after de novo assembly of callus mobilome-seq library and Tos17.
Fig 4.
Mobilome-seq detection of a novel active retrotransposon in rice seeds.
(A) Genome-wide analysis of mobilome-seq data identifies the PopRice retrotransposon family as the most represented active family in WT rice seeds. Legend as in Fig 3A. Pink dots indicate the windows corresponding to Osr4 and PopRice loci. (B) Detail of the depth of coverage of total mapped reads and split reads abundance of the O. sativa WT seeds mobilome-seq library at the PopRice locus on chromosome 2 (pink bar) for callus and leaf mobilome-seq data. Legend as in Fig 2C. (C) Phylogenic tree showing that PopRice is a distinct subfamily of Osr4 LTR-RT. The relative DOC calculated from two biological replicates in WT seed mobilome-seq data is indicated as a heatmap. (D) Dotter alignment of the scaffold #17 obtained after de novo assembly of WT seed mobilome-seq library and a PopRice element.
Fig 5.
PopRice retrotransposons produce extrachromosomal DNA during seed development in wild type rice.
(A) Southern blot experiment using non-digested genomic DNA extracted from WT rice leaves and seeds at different stages as indicated and detected with a PopRice specific probe (gDNA: genomic DNA, ecDNA: extrachromosomal DNA). The GelRed gel picture is shown as a loading control. (B) Southern blot experiment using non-digested genomic DNA extracted from dissected rice seed tissues as indicated and detected with a PopRice specific probe. Legend as in A. (C) Detection of PopRice circular forms using inverse PCR with primers localization depicted on the right (black bar: PopRice element, arrows: PCR primers, grey boxes: LTRs). Upper gel: PCR amplification of PopRice circles, middle gel: control PCR for PopRice detection, lower gel: PCR using eEF1α primers as control. (D) qRT-PCR analysis of PopRice and Osr4 transcripts in WT rice leaves, flowers and mature seeds. Two pairs of primers were used: PopRice specific primers (top) and primers specific for the whole Osr4 family (including PopRice) (bottom). The relative expression levels were calculated using eIF-5a as a reference, error bars indicate technical replicates, two biological replicates are shown for each tissue.