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Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion.

ABSTRACT: Targeted Genome Optimization (TGO) using site-specific nucleases to introduce a DNA double-strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)-mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre-existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.

SUBMITTER: Verkest A

PROVIDER: S-EPMC6576080 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion.

Verkest Aurine A Bourout Stephane S Debaveye Jurgen J Reynaert Kristine K Saey Bernadette B den Brande Ilse Van IV D'Halluin Kathleen K

Plant biotechnology journal 20190119 7

Targeted Genome Optimization (TGO) using site-specific nucleases to introduce a DNA double-strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)-mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre-existing transgenic locus in cotton. Here we describe the express ...[more]

PMID: 30549163

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Project description:BackgroundHeterosis is a complex phenomenon wherein the hybrids outperform their parents. Understanding the underlying molecular mechanism by which hybridization leads to higher yields in allopolyploid cotton is critical for effective breeding programs. Here, we integrated DNA methylation, transcriptomes, and small RNA profiles to comprehend the genetic and molecular basis of heterosis in allopolyploid cotton at three developmental stages.ResultsTranscriptome analysis revealed that numerous DEGs responsive to phytohormones (auxin and salicylic acid) were drastically altered in F1 hybrid compared to the parental lines. DEGs involved in energy metabolism and plant growth were upregulated, whereas DEGs related to basal defense were downregulated. Differences in homoeologous gene expression in F1 hybrid were greatly reduced after hybridization, suggesting that higher levels of parental expression have a vital role in heterosis. Small RNAome and methylome studies showed that the degree of DNA methylation in hybrid is higher when compared to the parents. A substantial number of allele-specific expression genes were found to be strongly regulated by CG allele-specific methylation levels. The hybrid exhibited higher 24-nt-small RNA (siRNA) expression levels than the parents. The regions in the genome with increased levels of 24-nt-siRNA were chiefly related to genes and their flanking regulatory regions, demonstrating a possible effect of these molecules on gene expression. The transposable elements correlated with siRNA clusters in the F1 hybrid had higher methylation levels but lower expression levels, which suggest that these non-additively expressed siRNA clusters, reduced the activity of transposable elements through DNA methylation in the hybrid.ConclusionsThese multi-omics data provide insights into how changes in epigenetic mechanisms and gene expression patterns can lead to heterosis in allopolyploid cotton. This makes heterosis a viable tool in cotton breeding.

Project description:Seed germination is considered the beginning of the spermatophyte lifecycle, and it is a crucial stage in determining subsequent plant growth and development. Although many previous studies have found that melatonin can promote seed germination, the role of melatonin in cotton germination remains unexamined. The main objective of this study is the characterization of potential promotional effects of melatonin (at doses of 0, 10, 20, 50, 100 and 200 μM) on cotton seed germination. This experiment demonstrated that low concentrations of melatonin can promote germination, while high concentrations failed to promote germination and even inhibited germination. Together, these results indicate that a 20 μM melatonin treatment optimally promotes cotton seed germination. Compared with the control, germination potential (GP), germination rate (GR), and final fresh weight (FW) increased by 16.67%, 12.30%, and 4.81%, respectively. Although low concentrations of melatonin showed some improvement in vigor index (VI), germination index (GI), and mean germination time (MGT), these effects were not statistically significant. Antioxidant enzyme activity during seed germination was most prominent under the 20 μM melatonin treatment. Superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased by 10.37-59.73% and 17.79-47.68%, respectively, compared to the melatonin-free control. Malondialdehyde (MDA) content was reduced by 16.73-40.33%. Two important plant hormones in seed germination, abscisic acid (ABA) and gibberellins (GAs), were also studied. As melatonin concentration increased, ABA content in seeds decreased first and then increased, and GA3 content showed a diametrically opposite trend, in which the 20 μM melatonin treatment was optimal. The 20 μM melatonin treatment reduced ABA content in seeds by 42.13-51.68%, while the 20 μM melatonin treatment increased GA3 content in seeds to about 1.7-2.5 times that of seeds germinated without melatonin. This study provides new evidence suggesting that low concentrations of melatonin can promote cotton seed germination by increasing the activity of antioxidant enzymes, thereby reducing the accumulation of MDA and regulating plant hormones. This has clear applications for improving the germination rate of cotton seeds using melatonin.

Dataset Information

Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion.

Publications

Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion.

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