Comprehensive characterization of T-DNA integration induced chromosomal rearrangement in a birch T-DNA mutant

Abstract

Background: Integration of T-DNA into plant genomes via Agrobacterium may interrupt gene structure and generate numerous mutants. The T-DNA caused mutants are valuable materials for understanding T-DNA integration model in plant research. T-DNA integration in plants is complex and still largely unknown. In this work, we reported that multiple T-DNA fragments caused chromosomal translocation and deletion in a birch (Betula platyphylla × B. pendula) T-DNA mutant yl.

Results: We performed PacBio genome resequencing for yl and the result revealed that two ends of a T-DNA can be integrated into plant genome independently because the two ends can be linked to different chromosomes and cause chromosomal translocation. We also found that these T-DNA were connected into tandem fragment regardless of direction before integrating into plant genome. In addition, the integration of T-DNA in yl genome also caused several chromosomal fragments deletion. We then summarized three cases for T-DNA integration model in the yl genome. (1) A T-DNA fragment is linked to the two ends of a double-stranded break (DSB); (2) Only one end of a T-DNA fragment is linked to a DSB; (3) A T-DNA fragment is linked to the ends of different DSBs. All the observations in the yl genome supported the DSB repair model.

Conclusions: In this study, we showed a comprehensive genome analysis of a T-DNA mutant and provide a new insight into T-DNA integration in plants. These findings would be helpful for the analysis of T-DNA mutants with special phenotypes.

Keywords: Birch; Chromosomal rearrangement; Multiple T-DNA; T-DNA integration; T-DNA mutant.

Fig. 1

Phenotypes of the gametes and seeds in yl. a Isolation of a yl mutant from BpCCR1 transgenic lines. WT, wild type birch. C11 and yl were the BpCCR1 overexpression lines. b Phenotypes of male inflorescence in WT, C11 and yl. c Fluorescein diacetate staining for pollen viability in WT, C11 and yl. Scale bars = 100 μm. d Phenotypes of infructescences and seeds in cross-combinations of WT × WT, C11 × WT and yl × WT. e 1000-seed weight of WT × WT, C11 × WT and yl × WT seeds. Scale bars = 1 cm (left) and 1 mm (right). f Germination rate of WT × WT, C11 × WT and yl × WT seeds. ** indicated P value < 0.01 (t test). Error bars represent the SD of three independent experiments

Fig. 2

Schematic diagram of chromosomal rearrangement induced by T-DNA in yl. Chr2–3 (red), Chr8–1 (blue) and Chr8–3 (green) were translocated fragments. Chr2–2, Chr2–5 and Chr9–1 in diagonal stripe pattern were the deletion regions. T-DNA was marked in yellow

Fig. 3

Schematic diagram and verification of the new junctions induced by T-DNA integration in the yl genome. a Schematic diagram of the primers’ location around the new junctions. Letters a to o represent the new junctions in the yl genome. b Gel electrophoresis of PCR amplification products. M1, DNA Maker DL15000. M2, DNA Maker DL2000. A1, PCR with primer IS4-R2 and P9182. A2, PCR with primer P9028 and P6345. A3, PCR with primer P6008 and P3895. A4, PCR with primer P6345 and IS5-F2. B1, PCR with primer IS2-F and P3054. B2, PCR with primer P3054 and IS3-R1. C1, PCR with primer IS6-R2 and P6008. C2, PCR with P6345 and IS6-F. c Verification of the junction between Chr2–4 and Chr8–1. Gel electrophoresis showed PCR amplification products with primer TB-R and IS3-F. N. Negative control (water)

Fig. 4

Sanger sequencing of the new junctions a to n in the yl genome. a The connection between Chr8–3 and Chr9–2. b The connection between Chr2–3 and Chr8–2. c The connection between Chr11–1 and Chr11–2

Fig. 5

Deletion of chromosomal fragments during chromosomal translocations. a Visualized reads coverage of yl and WT around IS5 using Integrative Genome Viewer (IGV). b Visualized reads coverage of yl and WT around IS2 and TB using IGV. c Relative Depth of Chr2 with 1-Mb resolution in yl compared to WT

Fig. 6

Diagram illustration of T-DNA integration model in the yl genome. a Overview of the three T-DNA integration models in yl genome. b Schematic diagram of the three T-DNA integrations which caused chromosomal translocation and/or deletion in the yl genome. Step 1 to step 3 indicated three T-DNA integrated into the yl genome, independently. T-DNA were marked in yellow. Dashed lines indicate rearrangements and arrows represent the direction of chromosomal rearrangement fragments. Chr8–3 (green), Chr8–1(blue), Chr2–3 (red) were translocated fragments. Chr2–2, Chr2–5 and Chr9–1 in diagonal stripe pattern were the deletion fragments