Performing Department
(N/A)
Non Technical Summary
The use of synthetic nitrogen fertilizers was critical in the Green Revolution and increased food production worldwide. However, this dependency on nitrogen fertilizers has significant economic and environmental repercussions. To reduce our heavy reliance on synthetic fertilizers and improve the sustainability of agriculture, we need to look for alternative nitrogen sources such as biological nitrogen fixation. Crops like maize, which require significant nitrogen inputs, particularly need such alternatives.Our recent studies showed that tropical maize landraces grown by Indigenous communities in Southern Mexico could obtain 29%-82% of their nitrogen from the air by hosting nitrogen-fixing bacteria in a mucilage produced by aerial roots after rainfall. Unfortunately, these accessions are unsuitable for growth in the US Midwest or other temperate locations worldwide due to their photoperiod sensitivity, tall stature, and long life cycles.In this USDA AFRI project, we lay the groundwork necessary to introduce the traits responsible for efficient biological nitrogen fixation into elite maize accessions adapter to the US Midwest. Our objectives are to:(1) determine the nitrogen-fixing ability of parent lines and establish a correlation between nitrogen fixation and plant traits easy to evaluate, such as the number of nodes with aerial roots and the diameter of these roots; (2) identify genetic markers related to nitrogen fixation efficiency; (3) evaluate the nitrogen benefits and potential yield trade-offs (penalties) of this new trait under various environments.The maize lines generated in this proposal and the new cultivars that will derive from them could also be helpful for breeding programs worldwide to reduce the economic burden that purchasing fertilizer can add to small-holder farmers and reduce the negative environmentalimpact of excess fertilizer use.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Our primary goal is to improve the sustainability of agricultural systems in the United States (US), specifically in the production of food, feed, and biofuels. Our primary objective is to reduce the reliance on synthetic nitrogen fertilizers in maize cultivation nationwide. To achieve this, we intend to incorporate the aerial root nitrogen fixation trait, found in select tropical landraces, into elite materials already well-adapted to the Midwest region of the US. Our efforts are aimed at creating a sustainable and efficient agricultural system.Objective 1: Determine the nitrogen-fixing ability of parent lines and establish a correlation with "proxy traits". Measuring the amount of nitrogen from biological nitrogen fixation in plants can mostly be done in the field using 15N dilution or 15N natural abundance techniques. However, these methods are costly and time-consuming, making them impractical for genetic mapping and breeding and more suitable for validation. To facilitate high-throughput phenotyping, we have developed a hypothesis based on our preliminary data that suggests a correlation between the levels of biological nitrogen fixation on maize aerial roots and the diameter of these roots. Additionally, the number of nodes with aerial roots seems to correlate with the duration of nitrogen acquisition from the air. Once we have confirmed these correlations, we can use these easily measurable parameters to speed up our breeding efforts.Objective 2: Identify genetic markers associated with traits related to nitrogen fixation. Our team is developing genetic mapping populations segregating for aerial root nitrogen-fixation-related traits. To achieve this, we will make various crosses between inbred lines adapted to the US Midwest and three tropical landrace parents, generating recombinant inbred lines and doubled haploid populations. Our team will evaluate traits such as aerial root diameter, number of nodes with aerial roots, number of aerial roots per node, flowering time, stalk diameter, ear and plant height, and ear morphology. Combining these genetic and phenotypic data will support the genetic mapping of nitrogen fixation-related traits.Objective 3: Evaluate the nitrogen benefits and potential yield trade-offs across multiple environments. By comparing physiological indices, such as photosynthetic efficiency, and the yield of segregating populations for characteristics previously correlated with biological nitrogen fixation in soils with restricted fertilization, we will determine the potential savings in nitrogen using this trait in elite materials. We will gain insights into better management of agricultural systems to reap the benefits of this trait. However, we must also consider the development of more aerial roots and the resulting increase in mucilage production, which incurs significant energy costs and may affect the yield performance of these materials. Therefore, we will investigate any potential drawbacks or "penalties" and assess how this trait introgression could impact their yield potential.
Project Methods
The methods will be presented here according to each specific objective, which may include one or more experiments. After the description of how each essay will be conducted, we are going to explain the measurement system through which we can evaluate the success of that stage of the project:Objective 1 - Methods for Experiment 1: testing the correlation between maize aerial root diameter and the biologically fixed nitrogen level. Three accessions with thick aerial roots and two with thin aerial roots will be assessed in a 15N dilution experiment in the greenhouse to evaluate the relationship between root diameter and nitrogen fixation. Firstly, we will inoculate their seeds immediately before planting with diazotrophs. Whole plants will be sampled 8 weeks after planting when all accessions are expected to have aerial roots producing a decent amount of mucilage. Next, we will evaluate the 15N abundance in plant samples by Isotope Ratio Mass Spectrometry (IRMS), allowing us to calculate the percentage of N derived from the air (% Ndfa).Objective 1 - Methods for Experiment 2: testing the correlation between the number of nodes with aerial roots and the biologically-fixed nitrogen level. To evaluate this correlation, two accessions showing a small number of nodes with aerial roots and two with a higher number of them will be evaluated through a 15N dilution experiment in a greenhouse with the same management and inoculation practices described in the previous experiment. As we intend to assess the % Ndfa over time, leaf samples will be collected every two weeks between the stages when the maize plant presents six leaves with a fully developed leaf collar (V6) and when they emit the tassel completely (VT). Aware that the phenological cycle of landraces will be delayed, we will continue sampling all plants until the landraces have issued the tassel. At the flowering stage, whole plant samples will be collected again. Thus, samples will be analyzed for 15N abundance by IRMS to calculate the % Ndfa.Objective 2 - Methods for Experiment 1: developing recombinant populations segregating nitrogen-fixing related traits. This segregating population is being developed after crossing an inbred line adapted to the Midwest of the US with a landrace parent. After obtaining the F1 hybrids, the plants have been self-fertilized through single-seed descent to generate recombinant inbred lines. These materials will be further advanced through self-pollination in the 2024 summer and winter nurseries to reach high levels of homozygosity by the 2025 summer. Field evaluations regarding the phenotypic uniformity of those plants and, eventually, genetic similarity tests through DNA markers may help us to assess homozygosity.Objective 2 - Methods for Experiment 2: development of doubled haploid populations, which were induced from backcrosses whose parentals diverge significantly regarding proxy traits related to the biological nitrogen-fixation traits. Those backcrosses were done before the haploidy induction, the first step towards double haploid production technology, to make the progeny more agronomically adapted to U.S. growing conditions.Objective 2 - Methods for Experiment 3: phenotypic analysis of segregating populations. For this purpose, those three segregating populations will be planted at the West Madison Agricultural Research Station and the Hancock Agricultural Research Station in Wisconsin in the summers of 2025 and 2026 for phenotypic evaluation. We will also include an additional field site in Georgia at the J. Phil Campbell Research and Education Center to compare performance under different environmental conditions. With a total of ∼1200 plots per site per year, performing 15N dilution tests at this scale is prohibitively expensive. For this reason, to select the most contrasting lines, we will focus on nitrogen fixation-related traits, like root diameter, number of nodes with aerial roots, number of aerial roots per node, flowering time, stalk diameter, ear and plant height, and ear morphology.Objective 2 - Methods for Experiment 4: genetic mapping recombinant populations segregating for nitrogen-fixing related traits. The segregating populations will be genotyped using tunable genotyping-by-sequencing. By default, reads will be aligned to the maize reference genome, but since we are using exotic landraces, we will also test other genomes to check for any significantly better alignment that should be adopted. Genetic and phenotypic data will be used to map nitrogen fixation-related traits genetically. Flowering time will be added as a covariate since it influences many maize physiological traits, including aerial roots. We will map each family individually, but all three families will also be mapped jointly by scoring genetic loci based on which of the founder lines they came from. Candidate genes within each quantitative trait loci will be identified based on the literature, focusing on genes and pathways involved in root development. If any large-effect locus is identified, we may also detect promising genetic markers that could be used in marker-assisted breeding. However, validation of these markers falls outside our scope.Objective 3: Evaluate the nitrogen benefits and potential yield trade-offs of recombinant inbred lines across multiple environments. Due to their highly diverse phenology and agronomic characteristics, we cannot directly compare nitrogen benefits and yield between inbred lines adapted to the Midwest of the U.S., modern hybrids, and giant tropical landraces. To test the contribution of the trait in genotypes approximating modern hybrids, we will evaluate isohybrids made from contrasting segregated lines crossed to complimentary testers. The first step was to generate the recombinant lines obtained from nine crosses between three inbred lines adapted to the Midwest of the U.S. and three landrace populations. These parentals were previously determined to exhibit divergent aerial root phenotypes. After obtaining the hybrids for each cross, the most contrasting plants were selected and self-fertilized in each generation. We selected ten recombinant lines from each of those nine crosses, five with the highest performance and five with the lowest, totaling 90 genotypes. To attain the 90 isohybrids, each was crossed, on winter nurseries, with testers complementary to one of their parentals. Aiming to add negative controls for this trail, three additional lines were made between the three inbred lines adapted to the Midwest and their proper complementary testers since we expect them to have greatly reduced or no nitrogen-fixing associated phenotypes. Finally, these 93 isohybrids will be evaluated to check for any penalty associated with nitrogen-fixation traits. This will be done by comparing their yields under three fertilizer regimes. Since nitrogen fixation does not occur before the emergence of aerial roots, we will use the same amount of starter fertilizer for the three nitrogen levels so that nitrogen from the two restrictive treatments will be suppressed during side-dressing fertilizations. To ensure uniform access to diazotrophs, all the seeds will be inoculated with a suspension containing the bacteria.