| Insertional mutagenesis using T-DNA or transposable elements is an important tool in functional genomics and is well established in several crops, but has not been routinely used in potato (Solanum tuberosum ). The tobacco (Nicotiana tabacum) Tnt1 retrotransposon has been successfully transformed, with transposition activity, in heterologous species including Arabidopsis (Arabidopsis thaliana), Medicago (Medicago trunculata), lettuce (Lactuca sativa), and soybean (Glycine max). In this thesis, the application of Tnt1 as an insertional mutagen in potato has been investigated.;The Tnt1 retrotransposon was introduced into a highly homozygous and self-compatible clone, M6, of the diploid wild potato species Solanum chacoense using Agrobacterium tumefaciens transformation. Seventeen independent transgenic lines (T0) were generated. Southern blot analysis demonstrated that all T0 plants contained at least one copy of transgenic Tnt1, and some transgenic Tnt1 transposed immediately after integration into the M6 genome. Subsequently, T0 plants were subjected to in vitro regeneration, which successfully induced retransposition of Tnt1 elements.;Insertional mutagenesis techniques utilize self-pollination to create homozygous insertional mutants. Therefore, the stability of the inserted mutagen during sexual reproduction is crucial to maintain the mutation. The stability of Tnt1 insertions in the potato genome was investigated in 80 selfed progeny derived from 4 different Tnt1-containing M6 lines, and it was determined that Tnt1 insertions were stable during sexual reproduction.;To identify the genomic positions of Tnt1 insertions, Tnt1-seq was developed. A total of 1,667 insertion sites from 70 Tnt1-containing lines were identified and showed Tnt1 preferentially inserted into genic regions. In addition, greenhouse phenotypic screening was performed on a total of 38 families derived from Tnt1 -containing lines. Several distinct phenotypes associated with plant stature and leaf morphology were discovered whereas progeny from wild type M6 did not show phenotypic variation. It is demonstrated that the insertional mutagenesis system based on Tnt1 and the M6 clone can be expanded to the genome-wide level to potentially tag every gene in the potato genome. |
