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SUMMARY Medicago truncatulais a model legume for fundamental research on legume biology and symbiotic nitrogen fixation.Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants inM. truncatulaR108. Approximately 21 000 insertion lines have been generated and publicly available.Tnt1retro‐transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE ofM. truncatulaR108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2‐kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site ofTnt1insertions inM. truncatulaR108 and stronger hypermethylation of genes correlated with higher number ofTnt1insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and theTnt1retrotransposition correlates with the hyperactive methylation regions.
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Genome‐wide analysis of flanking sequences reveals that Tnt1 insertion is positively correlated with gene methylation in Medicago truncatula
Summary From a single transgenic line harboring fiveTnt1transposon insertions, we generated a near‐saturated insertion population inMedicago truncatula. Using thermal asymmetric interlaced‐polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population.FSTrecovery from 14Tnt1lines using the whole‐genome sequencing (WGS) and/orTnt1‐capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference ofTnt1insertions showed thatTnt1is overall randomly distributed throughout theM. truncatulagenome. At the chromosomal level,Tnt1insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with theGCcontent. Based on 174 546 filteredFSTs that show exact insertion locations in theM. truncatulagenome version 4.0 (Mt4.0), 0.44Tnt1insertions occurred per kb, and 19 583 genes containedTnt1with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed thatTnt1‐inserted genes are significantly enriched in processes associated with ‘stress’, ‘transport’, ‘signaling’ and ‘stimulus response’. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583Tnt1‐inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide‐binding site‐leucine‐rich repeat genes harbored at least oneTnt1insertion, compared with the overall 38% ofTnt1‐inserted genes out of 50 894 annotated genes in the genome.
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- Award ID(s):
- 1733470
- PAR ID:
- 10460532
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 98
- Issue:
- 6
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 1106-1119
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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