Methods and systems for predicting the risk of transgene silencing
The present invention is based on the use of the H3K9me2 methylation levels in plant genomes to predict transgene silencing, transgene stability, and/or transgene expression level. Provided are methods and/or systems for generating whole-genome H3K9me2 maps and its use with an assigned threshold value for predicting gene silencing. The methods and/or systems provided herein can be used in high-throughput setting for screening large number of transformed event in a relatively short period of time as compared to existing technologies.
1. A method for predicting the risk of transgene silencing, comprising,
(a) generating histone methylation/acetylation profile data of a plant's genome from a physical assay;
(b) assembling the histone methylation/acetylation profile data of step (a) into a histone methylation/acetylation database showing peak numbers of the histone methylation/acetylation profiles;
(c) analyzing sequences of at least one transgene insertion site from a transgenic event;
(d) comparing the sequences of the transgene insertion site of step (c) with the histone methylation/acetylation database of step (b); wherein
(i) if the transgene insertion site falls into peak zero or peak one of the histone methylation/acetylation profiles, there is no risk of transgene silencing, or
(ii) if the transgene insertion site falls into peak two or more of the histone methylation/acetylation profiles, there is significant risk of transgene silencing;
(e) selecting the transgenic event with a lower probability of transgene silencing; and
(f) obtaining a plant that comprises the selected transgenic event.
2. The method of claim 1 , wherein the histone methylation/acetylation profile data is generated using a chromatin immunoprecipitation sequencing (ChIP-seq) assay.
3. The method of claim 1 , wherein the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations thereof.
4. The method of claim 1 , wherein the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3, and combinations thereof.
5. The method of claim 1 , wherein the histone methylation/acetylation profile data is associated with H3K9me2 methylation.
6. The method of claim 1 , wherein the plant is selected from soybean, maize, canola, cotton, wheat, and rice.
7. The method of claim 1 , wherein the sequences of the transgene insertion site are obtained by direct sequencing.