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EDTA.pl-genome -species others -step all -u -sensitive 0 -anno 1 -threads 48./p>99.9% for all replicates (three replicates from each LAB and QLD). The cytosine methylation level was calculated using the bismark_methylation_extractor in Bismark (v.0.19.0). The methylation ratio of cytosine was calculated as the number of methylated cytosines divided by the number of reads covering that position./p>0.5 TPM was used for downstream analysis. Values of this combined analysis were used to determine the relative expression of homeologues. The homoeologous pairs were defined as expressed when the sum of the a and b subgenome homeologues was >0.5 TPM. This filtration included duplicate pairs in which only a single homeologue was expressed. To standardize the relative expression of homeologues, the absolute TPM for each gene within the duplicate pair was normalized as follows. A and B represent the genes corresponding to the A and B homeologues in pairs./p>95%) and >50% coverage were identified as integrated T-DNA in the plant genome. For the stable transformation analysis, leaf tissues were collected from 5-week-old N. benthamiana stable transgenic independent lines generated using pFN117 (Cas9) and pUQC-GFP-(218). Genomic DNA was extracted following the cetyltrimethylammonium bromide method. Nested, insertion-specific primers for the right borders (RB1, RB2 and RB3 RB2 and RB3; Table 2) of pFN117 and pUQC-GFP-(218)-A were designed. Arbitrary degenerate primers and the high-throughput thermal asymmetric interlaced polymerase chain reaction (ht-TAIL-PCR) program were as described by Singer and Burke93. Purified PCR products were directly Sanger sequenced using RB3 primer, and the insertion sites were identified through a BLASTn search against the N. benthamiana genome. The number of stable and transient T-DNA insertion sites that intersect gene body, promoter, terminator and TEs were determined using the bedtools Intersect tool (v.2.30.0)92 and the length to the closest gene from the insertion site was calculated using RnaChipIntegrator (v.1.1.0) (https://github.com/fls-bioinformatics-core/RnaChipIntegrator). The z-score test for two population proportions was used to determine the significant difference between 10 kb, 10–20 kb, 20–30 kb and 30–40 kb intervals from all stable, transient transgene insertion sites and randomly selected sites in the N. benthamiana genome./p>

15kbp and surrounding syntenic genes in are shown in orange, purple, yellow and brown. Orthology/homology relationships are indicated by coloured shading. In (B), distances between genes indicated (black text)< 50kbp; (red text) >50kbp. TE annotation tracks for LAB and QLD were prepared using annotation data from the EDTA TE annotation pipeline (see online Methods) and Geneious Prime software (Geneious Prime 2023.0.1; https://www.geneious.com). Only LTR-transposable elements are shown. Yellow blocks represent GYPSY elements and green blocks represent COPIA elements. The size of each block is proportional to the number of base-pairs annotated for that element. Red triangles represent LTR repeat regions that flank either a GYPSY or COPIA element. These elements are likely to be nearly complete and can be considered possible autonomous elements. The rectangular red blocks flank unknown LTR-TE elements. Unknown TEs are elements that are recognized as an LTR element but are not able to be classified as either a COPIA or GYPSY element due to irregularities in internal sequences for that element. These are likely to represent non-autonomous elements. Those elements not flanked by LTR sequences are highly fragmented nonfunctional elements. The blue rectangular boxes highlight the location of the genes annotated in the tracks above and below the TE annotation tracks./p>