Identifying genes related to symbiotic nitrogen fixation in Medicago truncatula Tnt1 insertion mutants using a forward genetics approach
Date
2020
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Publisher
University of Delaware
Abstract
Nitrogen is an essential nutrient required by all plants. Although the earth’s atmosphere consists of approximately 78% nitrogen, it exists in an unusable form for plants. Current agricultural practices rely on industrial nitrogen fixation to produce fertilizers that improve crop productivity. However, this method requires the use of precious natural resources and can cause a variety of negative impacts on the environment and human health. Biologically fixed nitrogen is a favorable alternative accessible to leguminous plants through their symbiotic relationship with soil bacteria called rhizobia. Genetic studies aimed at identifying plant genes related to successful nodulation and nitrogen fixation are underway to help better understand this infection process and improve the performance of legumes in agriculture. To date, over 21,000 Tnt1 mutant lines of Medicago truncatula, a model legume used for genetic research, have been created. Several lines displaying defects in symbiotic nitrogen fixation (SNF) have been identified and are referred to as Fix- mutants. In this research, segregation analysis of the Fix- mutant NF18598 indicated loss of SNF function could be governed by a dominant mutation. A total of 114 Tnt1 insertions were identified in the NF18598 genome using three different sequencing methods. Tnt1-capture sequencing (SC) found the largest number of insertions (70) while the other two methods, thermal asymmetric interlaced PCR (TAIL-PCR) and whole genome sequencing (WGS), found a similar number of insertions (40 and 41, respectively). It was found that 50% of the insertions were located in coding regions. One of the Tnt1 insertions located on chromosome 5 is found within the exon of a phosphatase 2C family protein gene (Medtr5g009370) and is upregulated in nodules. While there was a significant SNF association with Medtr5g009370 in the R1 generation, this association was not detected in the R2 generation. Light microscopy analysis of nodules displayed clear differences between mutant samples identified as either Fix+ or Fix-. The Fix- mutant nodules displayed some cellular organization but distinct developmental zones could not be identified. Infection threads were observed in Fix- nodules that appeared to contain bacteria that had not been released; however, further microscopy analysis must be conducted to verify the presence of rhizobia. In addition, further research involving a larger sample population would help verify if the insertion located in Medtr5g009370 is responsible for the defective nodule phenotype observed and if the phosphatase gene is involved in symbiotic nitrogen fixation.
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Keywords
Nitrogen fixation, Medicago, Nodule, Rhizobia, Symbiosis, Tnt1, Agricultural practices, Legumes