Identifying Tnt1 transposon insertions in the NPF1B gene of Medicago truncatula through PCR screening

The NPF1B gene is retained in many species that are able to perform root symbioses, mycorrhizae and symbiotic nitrogen fixation, and lost in those that don’t, but its function is unknown. NPF1B is part of a clade of genes in the Nitrate and Peptide transporter Family of genes. Within its subfamily there are five clades (A, B, C, D1, and D2), with MtNPF1B found in the B clade. While work has been done to characterize genes in the C clade in Medicago truncatula as well as the B and C clades in Lotus japonicus, it will be informative to understand the role of the B gene in Medicago in the context of the bigger picture, in terms of its role in the evolution of the nitrogen fixation symbiosis, a vital symbiotic relationship between legumes and Rhizobium bacteria. Since the B gene is closely related to the C gene, which has a characterized lateral root and nodule phenotype, I want to determine if the B gene shares a function with the C gene, or if it has a unique role in Medicago. In order to study the MtNPF1B gene function, potential transposon insertions were identified from a public database. I am working to identify and locate these Tnt1 transposon insertions in the NPF1B gene in R108 background Medicago. Using gene and transposon-specific primers, I am using PCR to screen for insertions within the gene of interest. The goal is to identify plants that are homozygous for a mutation in the NPF1B gene, which will allow for further study of a potential mutant phenotype as a result of the transposon mutagenesis. Characterizing the NPF1B gene is of particular interest because it will provide insight into the gene’s role in root formation, nodulation, and symbiotic nitrogen fixation