Next-generation sequencing (NGS) approaches are attractive alternatives to the PCR-based characterisation of genetically modified plants for safety assessment and labelling since NGS is highly sensitive to the detection of T-DNA inserts as well as vector backbone sequences in transgenic plants. In this study, two independent transgenic male Populus tremula lines, T193-2 and T195-1, both carrying the FLOWERING LOCUS T gene from Arabidopsis thaliana under control of a heat-inducible promoter (pHSP::AtFT) and the non-transgenic control clone W52, were further characterised by NGS and third-generation sequencing. The results support previous findings that the T-DNA was hemizygously inserted in one genomic locus of each line. However, the T-DNA insertions consist of conglomerations of one or two T-DNA copies together with a small T-DNA fragment without AtFT parts. Based on NGS data, no additional T-DNA splinters or vector backbone sequences could be identified in the genome of the two transgenic lines. Seedlings derived from crosses between the pHSP::AtFT transgenic male parents and female wild type plants are therefore expected to be T-DNA splinter or vector backbone free. Thus, PCR analyses amplifying a partial T-DNA fragment with AtFT-specific primers are sufficient to determine whether the seedlings are transgenic or not. An analysis of 72 second generation-seedlings clearly showed that about 50% of them still reveal the presence of the T-DNA, confirming data already published. To prove if unanticipated genomic changes were induced by T-DNA integration, extended future studies using long-range sequencing technologies are required once a suitable chromosome-level P. tremula reference genome sequence is available.

中文翻译：

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两个转基因早开花杨树系的测序证实了无载体的单基因座T-DNA整合。

下一代测序（NGS）方法是遗传修饰植物基于PCR表征的安全评估和标记的有吸引力的替代方法，因为NGS对转基因植物中T-DNA插入片段以及载体主链序列的检测高度敏感。在这项研究中，两个独立的转基因雄性杨木系，T193-2和T195-1，均在热诱导型启动子（pHSP :: AtFT）和非转基因对照的控制下携带拟南芥的FLOWERING LOCUS T基因。 NGS和第三代测序进一步鉴定了W52克隆。该结果支持以前的发现，即将T-DNA半合子地插入每个株系的一个基因组基因座中。然而，T-DNA插入片段由一个或两个T-DNA拷贝的聚集体以及一个小的无AtFT部分的T-DNA片段组成。基于NGS数据，在两个转基因品系的基因组中不能鉴定出额外的T-DNA分裂或载体主链序列。因此，预计将从pHSP :: AtFT转基因雄性亲本和雌性野生型植物之间的杂交获得的幼苗不含T-DNA碎片或载体骨架。因此，用AtFT特异性引物扩增部分T-DNA片段的PCR分析足以确定幼苗是否是转基因的。对72个第二代幼苗的分析清楚地表明，其中约50％的幼苗仍显示T-DNA的存在，从而证实了已经发表的数据。为了证明T-DNA整合是否诱导了意料之外的基因组变化，