Sequencing of two transgenic early-flowering poplar lines confirmed vector-free single-locus T-DNA integration

• Published in: Transgenic research Vol. 29; no. 3; pp. 321 - 337
• Main Authors: Kersten, Birgit, Leite Montalvão, Ana Paula, Hoenicka, Hans, Vettori, Cristina, Paffetti, Donatella, Fladung, Matthias
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-06-2020; Springer Nature B.V
• Subjects: Animal Genetics and Genomics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Deoxyribonucleic acid; DNA; DNA sequencing; FLOWERING LOCUS T gene; Genetic Engineering; Genomes; Integration; Labeling; Life Sciences; Molecular Medicine; Next-generation sequencing; Nucleotide sequence; Original Paper; Plant Genetics and Genomics; Polymerase chain reaction; Populus tremula; Seedlings; T-DNA; Transgenic plants; Transgenics; Early-flowering; Poplar breeding; Transgene-free; Biosafety research; Populus
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-020-00203-0

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.