Genome‐wide analysis of flanking sequences reveals that Tnt1 insertion is positively correlated with gene methylation in Medicago truncatula

Abstract

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago 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. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-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 of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1inserted 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 19 583 Tnt1-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 one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element

“…In order to verify the active transposition of Tnt1 in B. distachyon R1 lines, we used three different methods to identify the transposition events: thermal asymmetric interlaced-PCR (TAIL-PCR), whole-genome sequencing (WGS) and sequence capture (Sun et al, 2019). We used TAIL-PCR to amplify the PCR products using Tnt1 LTR specific primers and arbitrary degenerate primers as described previously (Singer and Burke, 2003;Cheng et al, 2011;Cheng et al, 2014;Cheng et al, 2017).…”

“…Some of the insertions were found in the coding regions of B. distachyon genes (Table S1). From our previous analyses of FSTs in M. truncatula Tnt1 insertion lines we know that TAIL-PCR can recover <50% of total FSTs in any given line (Sun et al, 2019). Encouraged by the TAIL-PCR results for B. distachyon, we sought to identify most of the insertions in all the R1 lines by taking advantage of two methodologies, sequence capture (Sun et al, 2019) and WGS (Veerappan et al, 2016).…”

“…From our previous analyses of FSTs in M. truncatula Tnt1 insertion lines we know that TAIL-PCR can recover <50% of total FSTs in any given line (Sun et al, 2019). Encouraged by the TAIL-PCR results for B. distachyon, we sought to identify most of the insertions in all the R1 lines by taking advantage of two methodologies, sequence capture (Sun et al, 2019) and WGS (Veerappan et al, 2016). For WGS, DNA samples were extracted from the whole leaves of B. distachyon R0 mother plants (MP2 and MP15) and R1 (nc15-006, nc15-007 and nc15-008) plants using the Qiagen DNA extraction kit according to the manufacturer's instructions.…”

“…Since WGS is expensive and time-consuming, we decided to use the sequence capture method (Sun et al, 2019) to isolate most FSTs in a given line. We identified a total of 76 Tnt1 transposition events in 16 R1 lines tested, with an average insertion of 4.75.…”

“…In contrast, retrotransposon mutagenesis allows multiple insertions per plant. For example, it has been reported that there is an average of 80 insertions per line in M. truncatula (Tadege et al, 2008;Sun et al, 2019), from 4 to 19 insertions per line in soybean (Cui et al, 2013) and 24 insertions per line in potato (Duangpan et al, 2013). The high efficiency of transposition of class 1 elements is due in part to its 'copy and paste' mechanism (Kumar and Bennetzen, 1999), while class 2 elements have a 'cut and paste' mechanism (Wessler, 2006).…”