The position and complex genomic architecture of plant T-DNA insertions revealed by 4C

The integration of T-DNA to plant genomes is widely used for basic research and agriculture. High heterogeneity in the number of integration events per genome, their configuration and impact on genome integrity highlight both, the critical need and great challenge to detect the genomic locations of T-DNA insertions and their associated chromosomal rearrangements. Here we present '4SEE', a circular chromosome conformation capture (4C) based method for robust, rapid and cost-efficient detection of the entire scope T-DNA locations. Moreover, by measuring the chromosomal architecture at plant genome flanking the T-DNA insertions, 4SEE outlines their associated complex chromosomal aberrations. Applying 4SEE to a collection of confirmed T-DNA lines revealed previously unmapped T-DNA insertions and chromosomal rearrangements such as inversions and translocations. Uncovering such events in feasible, robust and cost-effective manners by 4SEE in any plant of interest have implications for accurate annotation and phenotypic characterization of T-DNA insertion mutants and transgene expression in basic science applications as well as for plant biotechnology. Overall design: 4C-seq was performed on WT, SALK_011436, SALK_009469, SALK_041474, SALK_064627, SALK_005512 from the SALK stock, and CS68932 from the Arabidopsis Biological Resource Center (ABRC) were used in this study. There are 118 samples, as follows: three endogenous genes (two replicas each), four SALK lines in a gradient of template amounts: six samples of SALK_063720 and SALK_064627, five samples of SALK_011436 and seven samples of SALK_005512. The remaining SALK lines have 1 sample each. Seventy-two files are on the ADF viewpoint (CS68932), with a gradient of template amounts and reads. The remaining 14 samples are seven viewpoints measured in WT or CS68932 plant, which map the chromosomal rearrangements.

转移DNA（T-DNA）整合至植物基因组的技术，广泛应用于基础研究与农业领域。单个基因组中T-DNA整合事件的数量、整合构型及其对基因组完整性的影响存在高度异质性，这既凸显了精准检测T-DNA插入位点及其伴随染色体重排的迫切需求，也带来了巨大挑战。本研究开发了"4SEE"技术——一种基于环状染色体构象捕获（circular chromosome conformation capture, 4C）的方法，可稳健、快速且经济高效地检测全范围T-DNA插入位点。此外，通过分析T-DNA插入位点侧翼的植物基因组染色体架构，"4SEE"还能揭示其伴随的复杂染色体畸变。将"4SEE"应用于已验证的T-DNA突变体集合后，研究人员发现了此前未被定位的T-DNA插入事件与染色体重排（如倒位和易位）。凭借操作简便、结果可靠且成本低廉的优势，"4SEE"可在任意目标植物中揭示此类事件，这对于T-DNA插入突变体的精准注释与表型表征、基础研究中转基因表达分析，以及植物生物技术应用均具有重要意义。

整体实验设计：本研究对野生型（WT）、SALK种质库的SALK_011436、SALK_009469、SALK_041474、SALK_064627、SALK_005512，以及拟南芥生物资源中心（Arabidopsis Biological Resource Center, ABRC）保藏的CS68932开展了4C-seq实验。本次实验共包含118个样本，具体如下：3个内源基因（每个设置2个生物学重复）；4个SALK突变体按模板量梯度设置样本：SALK_063720与SALK_064627各6个样本，SALK_011436共5个样本，SALK_005512共7个样本；其余SALK突变体各设1个样本。其中72个样本基于ADF视角（CS68932）构建，覆盖了不同的模板量与测序读长梯度。剩余14个样本则分别在野生型或CS68932植株中开展了7个视角的测序，用于定位染色体重排事件。