Progress 06/01/24 to 05/31/25
Outputs
Target Audience:Among the seven groups that form our initial target audience (corn processing industry, corn growers, scientific community, postdocs, graduate students, undergraduate students, and K-12 students), only the first group was not reached by our efforts during this reporting period, as the corn industry will benefit in the medium/long term. The others benefited as follows: Cron growers have already had their "first contact" through a field day (listed later in this report, in the section dealing with "dissemination of results to the community of interest") with some corn hybrids obtained in our breeding program that aims to introgress the nitrogen fixation-related traits based on aerial root phenotypes from tropical maize landraces into selected elite materials. It is worth mentioning that these hybrids are NOT those that can be future launched on the corn seed market since their parents are not yet recombinant inbred lines (RILs); that is, they are not yet homozygous and, therefore, need to be advanced a few more generations. Also, as everyone may know, a series of technical and legal provisions precede the launch of a new hybrid on the market. Still, corn growers were able to see firsthand the huge difference in plant vigor and yield-associated indexes between hybrids with and without the introgressed aerial root-related trait: when comparing hybrids with similar genetic backgrounds, those with aerial roots produced 65-90% more under conditions of severe nitrogen deficiency (-80 points of nitrogen per acre). These results, which are exposed in more detail in the "Accomplishments" section, have left many growers anxious about the future availability of this technology. The scientific community, particularly researchers interested in biological nitrogen fixation, was reached through the conferences our team attended, also listed in the "Productions" section. During such conferences, we have received excellent feedback from colleagues, who highlighted that the understanding (in genomic/molecular terms) and the introgression of traits linked to biological nitrogen fixation in grasses would be a major game changer. The postdoc working in the program, Dr. Rubens Diogo, from Ané Lab at the University of Wisconsin-Madison), benefited from the training he received to perform the activities inherent to this project, especially concerning the techniques involved in 15N isotope dilution, gene mapping, and management of agricultural systems, which form the core of its three main goals. Graduate students from the PI and co-PIs' laboratories benefited from observing/participating in the activities carried out during the project and from weekly meetings of the respective research groups, in which details about the techniques adopted in this project and the results achieved have been constantly shared. As an example, a graduate student from Ané Lab, Valentina Infante, who has proven experience in microbiology, has actively participated in the preparation of diazotrophic colonies that were sprayed on the plants of our field trials implemented in Hancock, WI (experiment detailed in the "Accomplishments" section). The undergraduates were one of the groups that benefited most from the activities carried out during this reporting period since those involved in our project were able to closely follow (some for the first time) a research work while also receiving instructions on why each activity was performed, something that, according to their own testimony, was of great value in understanding several subjects with which they had only had theoretical contact during their respective classes. It is worth noting that many of them, whose names are presented later (in the section on "opportunities for training and professional development"), were paid hourly to work in our corn fields and later, during drying, threshing, grading, processing, and organizing the seeds. This compensation helped many of them with their housing and meal commitments. Lastly, K-12 students were reached through several events, such as field days and fairs aimed at this audience and their respective families. Among these events, we highlight the UW Science Expeditions (held on April 4th to 6th, 2025) and the UW Family Gardening Day (May 3rd, 2025), both hosted by the University of Wisconsin-Madison. At these events, the techniques applied in our project and the results obtained are explained in a didactic manner appropriate to these young people's age and education level. Many of them had shown a particular fascination with the stuff we had made available (laboratory equipment, microscope samples, exotic plants, etc.). At the same time, some said they would like to pursue a research career in the future. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In recent months, the project has funded a postdoctoral researcher and eleven undergraduates who work/worked on the team at Dr. Jean-Michel Ané's laboratory at the University of Wisconsin. Dr. Rubens Diogo is a postdoctoral researcher on the maize genetics of nitrogen fixation on aerial roots. An agronomist with experience in corn breeding, biotechnology, and seed production, Dr. Diogo has received solid training in microbiology (especially concerning the physiology of diazotrophs), biochemistry (biogeochemical cycles of nitrogen), and bioinformatics (use of various tools for interpretation of genomic and sequencing data) on recent months while working in this project. In addition to attending frequent seminars hosted by the University of Wisconsin-Madison, such as the "R Programming Workshops Series" (https://researchguides.library.wisc.edu/R), and the seminars organized by the Center for Genomic Science Innovation (https://cgsi.wisc.edu/seminars/) and the Plant Cellular and Molecular Biology supergroup (https://plantcmb.wisc.edu/events/category/plantcmb-weekly-seminar/), Dr. Diogo has participated in weekly meetings of the research group led by Dr. Jean-Michel Ané, where teammates (members involved in both plant and microbial research) share their knowledge and results. In addition, he has participated in the monthly meetings held by the NFARM (Nitrogen-Fixation on the Aerial Roots of Maize) team (https://nfarm.bact.wisc.edu/about-us/), a research group focused precisely on conducting this project and other related ones, as well as in the monthly meetings of soNAR (sorghum Nitrogen-fixation on aerial roots) team (https://sonar.bact.wisc.edu/about-us/), another research group that carries out studies similar but focused on sorghum crop. Earlier this year, Dr. Diogo even had the opportunity to attend his first Plant and Animal Genome Conference, the PAG32 (https://intlpag.org/PAG32), between January 10th and 15th, 2025, in San Diego, California. Among more than 200 scientific workshop sessions available there (https://plan.core-apps.com/pag32/events), he has participated in lectures at the following workshops: "Mutations", "Genomics of seed quality traits", "Systems biology and ontologies", and "Integration of functional genomics data in genetic and genomic research" (on January 10th), "Allele mining", "Plant nitrogen use efficiency engineering", "Plant molecular breeding", and "Maize" (on January 11th), "Gene introgression", "Climate-smart agriculture", "Transposable elements", and "Functional genomics" (on January 12th), "Application of genetics and genomics for large-scale breeding programs", "Integrative approaches for scaling trait discovery in crop plants", and "Genomics-assisted breeding in hybrid crops for global food security" (on January 13th), "Genomics-assisted breeding", "Root nodule symbiosis", "Plant cistromics", and "Phenotypic plasticity and genotype-by-environment interaction" (on January 14th). All these meetings, workshops, and conferences attended in recent months were of great value to Dr. Diogo for improving his knowledge in the areas where he works as a researcher. During the last months, eleven undergraduates also worked on the fields we conducted in Verona-WI and Hancock-WI: Caelin Smith, Madigan Freng, McKenna Riley, Jason Pan, Ember Foreman, Damien Zingaro, Swayamshuvra Chakraborty, Ariff Ismadi, Andi Koslowski, Isabelle Koo, and Elliot Patrenets. They all received training in the following areas: management of corn fields (from planting to harvest), techniques for manually pollinating corn, basic principles of corn breeding, cultivation of diazotroph colonies, and organization of germplasm banks. In addition to following the step-by-step process of conducting the experiments, they actively participated in collecting the results when they were instructed on the importance of fulfilling all the essential criteria of scientific methodology. They also participated in the laboratory members' meetings, where they were able to learn about the projects conducted by each researcher and, thus, have a more holistic view of the plant-microbe symbiosis theme. One of these students, Caelin Smith, who had an outstanding performance in the activities of this project and graduated at the end of last year, ended up being hired by Ané Lab as a Research Intern and is currently working on another project developed by Jean-Michel Ané's team. How have the results been disseminated to communities of interest?The results observed in the experiments carried out so far are being shared in different ways, such as lectures at conferences and workshops (listed in the "Products" sections), publications that include conference papers and a scientific article in preparation for submission to a peer-reviewed journal, and events for the community at large, which include field days at the stations where the experiments were conducted (Hancock-WI, Verona-WI, and Watkinsville-GA) and science fairs such as UW Science Expeditions and UW Family Gardening Day. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Determine the nitrogen-fixing ability of parent lines and establish a correlation with "proxy traits". Regarding the first goal (correlation between nitrogen fixation-related trait and "proxy traits", that is, the aerial root-based phenotype), we intend to perform new 15N isotope dilution assays, this time using plant populations with different genetic backgrounds, but preferably F2 populations originating from specific crosses between Midwest-adapted elite inbreds and tropical maize landraces.Currently, in our germplasm bank, we have F2 seeds from 17 crosses that could potentially be used for this type of study. The choice of the F2 population(s) with which we will carry out the new 15N isotope dilution assays depends on a series of variables that we are still considering, such as plant height (since the experiments are all carried out in greenhouses), duration of their phenological cycle, and, mainly, aspects linked to nitrogen fixation-related traits based on aerial root phenotype, such as phenotypic differences between the parents and range of variability across the F2 individuals.After superimposing the results of all these trials involving the 15N isotope dilution technique and aerial root-based phenotyping, we can more precisely establish a correlation between them. Goal 2: Identify genetic markers associated with traits related to nitrogen fixation. Regarding the second goal (genetic markers associated with nitrogen fixation-related traits based on aerial root phenotypes), eight doubled haploid populations whose genotyping data are already available will be phenotyped in Verona-Wisconsin during the summer of 2025. Two of these populations, which were mentioned above (in the "Accomplishments" section), will be phenotyped for the third consecutive year, while one of them will be phenotyped for the second year. The other five double-haploid populations will be phenotyped for the first time. In total, 1,386 entries (which make up these eight populations) will be phenotyped; considering two replicates and six samples per plot, 16,632 plants will have their nodes and aerial roots measured, and agronomic traits of utmost importance.Still concerning the second goal, we intend to perform a bulked segregant analysis (a genetic mapping technique that compares DNA pools from individuals with contrasting phenotypes from a segregating population) on some F2 populations originating from crossing tropical maize landraces into selected elite materials. As mentioned in the previous item, we already have F2 seeds from 17 populations in our germplasm bank, which may be used for this purpose. Our objective is to use the results of these analyses to narrow down the previously identified QTL, helping us to prospect promising genes for nitrogen fixation-related traits based on aerial root phenotype.We aim to fine-tunetheseQTL as much as possible. We are already setting up a new series of experiments to verify the gene expression on nodes that form aerial roots in certain tropical maize landraces. In these experiments, we will isolate tissue from the nodes of Oaxa524 landrace plants at various stages of aerial root formation, from lower nodes with well-developed aerial roots to upper nodes without any sign of pre-development of these roots. After isolating the respective tissues from these nodes, we will extract the RNA and submit it to RNA sequencing (RNA-Seq) analysis. The objective will be to ascertain which genes, especially those in the QTL we previously identified, have a more significant differential expression during the different stages of aerial root development. Once identified, these genes can be better studied in the future through gene overexpression versus gene knock-out techniques.Finally, regarding the aforementioned "edge effect" and the possible influence of light quality on this phenomenon, we intend to conduct a trial under greenhouse conditions involving a deliberate distortion of the red to far-red light ratio (R:FR). Initially, we want to check whether the decrease in this ratio, which triggers a "shade avoidance response" (SAS, a condition in which plants in very dense cultivation are subject in the field), would cause, in addition to stem elongation and increase of leaf area (well-studied consequences of SAS), also a significant decrease in the number of nodes and/or the aerial roots diameter in genotypes that naturally express this trait in a very pronounced way, such as the Oaxa524 landrace. Depending on the results obtained, other light experiments will be conducted in our segregating maize populations for nitrogen fixation-related traits based on the aerial root phenotype. Goal 3: Evaluate the nitrogen benefits and potential yield trade-offs across multiple environments. Following the promising results achieved in 2024, we will conduct a much larger trial during the summer of 2025. In this trial, in addition to the nine hybrids previously tested (which will be assessed for the second consecutive year), we will evaluate another 150 hybrids recently produced in our winter nursery. To design these hybrids, we chose 30 double haploid lines contrasting for nitrogen fixation-related traits based on aerial root phenotypes. It means that, within the range of variability observed in their respective populations, these 30 lines consistently presented extreme phenotypic values concerning the number of nodes with aerial roots and the diameter of these aerial roots. These 30 lines were then crossed with five testers (01DIB2, NP948, PH44A, HC33, and LH244) well adapted to the Midwest's growing conditions.All these hybrids will be grown under two different soil conditions (standard nitrogen fertilization and severe nitrogen deficiency) and inoculated with diazotrophs. Two replicates will be adopted for each treatment. Thus, they will be evaluated for several variables, including important agronomic traits (flowering time, ear and plant height, stalk diameter, root and stalk lodging propensity), all relevant aerial root-related traits (number of nodes with aerial roots, number of aerial roots per node, and aerial roots diameter) and, mainly, yield-related (grain productivity, test weight, grain moisture, and thousand-grain weight) and nitrogen status-related traits, like chlorophyll content, which will be measured in two phenological stages (R1 and R3, i.e., silking and milk stages). The plots will be harvested using a two-row combine that can collect grain samples. Grain samples from hybrids with more contrasting values will still be subjected to nitrogen and carbohydrate (starch plus reducing sugars) content analyses.
Impacts
What was accomplished under these goals?
Goal 1: Determine the nitrogen-fixing ability of parent lines and establish a correlation with "proxy traits". During these last months, we conducted two bifactorial trials involving the 15N isotope dilution method under greenhouse conditions. This technique, adopted to quantify nitrogen fixation in plants through isotopically labeled fertilizers added to pots, allows the tracking of plant nitrogen absorbed from the soil solution, enabling the distinction between nitrogen percentages coming from the fertilizer and fixed from the atmosphere.In the first of these trials, we tested two genotypes (a Midwest-adapted elite inbred, PHP02, and a tropical maize landrace, Oaxa524), whose plants (15 per treatment, 60 in total) were inoculated with bacteria from one of the two following groups: diazotrophic or non-fixing. In each of the 60 plants that comprised the experiment, we assessed the atom% 15N excess, a parameter used to calculate the percentage of nitrogen derived from the atmosphere (%Ndfa). This experiment revealed that the tropical maize landrace, which exhibits the aerial root-related traits (i.e., has a much larger number of nodes with thick aerial roots), fixes more than three times the amount of atmospheric nitrogen fixed by the elite inbred, which lacks that trait. Their %Ndfa was 50% and 15%, respectively.In the second experiment involving 15N isotope dilution, we adopted a methodology similar to the previous one. Nevertheless, we used other genotypes: segregating lines obtained after F1 hybrids (originated from a cross between Oaxa524 and PHP02) had been selfed or backcrossed. Furthermore, in this case, in addition to measuring the atom% 15N excess, we also phenotyped the plants for aerial root-related traits, which include the number of nodes with aerial roots, the number of aerial roots per node, and the diameter of those aerial roots. The results of this experiment are still being processed. After such processing, we can estimate a first correlation between the indexes of %Ndfa and aerial root-related traits, which are also continuous quantitative variables. Goal 2: Identify genetic markers associated with traits related to nitrogen fixation. Two populations of doubled haploid maize lines, previously developed and genotyped (using a chip with 1,800 markers) in collaboration with Limagrain, were phenotyped last year, as they had been in 2023, at two different locations: Verona-Wisconsin and Watkinsville-Georgia. These phenotypic data included aerial root-related traits (number of nodes with aerial roots, number of aerial roots per node, and aerial root diameter) and traits of primary agronomic importance (stalk diameter, plant height, and number of days to anthesis). After overlapping the genotypic and phenotypic data obtained with these two populations, we observed 16 unique quantitative trait loci (QTL) for aerial root-related traits spread across the 10 chromosomes of maize: four QTL associated with the number of nodes with aerial roots, three QTL for the number of aerial roots per node, and nine QTL for the aerial root diameter.However, most of the QTL we identified are large, probably because those double haploid plants were conceived from populations in which the F1 hybrid (obtained after the cross between a tropical maize landrace and a Midwest-adapted elite inbred) was backcrossed with the elite parent before the haploid induction. This fact resulted in a narrower range of genomic variation and, consequently, in a relatively minor number of possible allelic recombinations during the crossover events.Since then, these QTL have been constantly mined to identify genes associated with such aerial root-related traits. To this end, we are using several strategies, such as literature support (i.e., looking for information about genes present in the genome regions where the 16 QTL were observed), searching for orthologous genes (for example, those that have remarkable similarity, in terms of nucleotide sequences, with aerial root-related genes annotated in the genome of sorghum or other members of the Andropogoneae tribe), among other approaches.We also carried out, in Verona-WI, the phenotyping of a BC1S5 population originating from a cross between the elite inbred B73 and a tropical maize landrace. Researchers at Penn State University kindly provided seeds and genotyping data for this population. The mapping results generated with this population are also used to fine-tune our previously identified QTL.Regardless of the populations we deal with, we have observed a pronounced effect of the plant's position in the row on the expression of aerial root-related traits. Knowing that this phenomenon would greatly influence the heritability of the trait we are interested in, we decided to phenotype all the plants in the breeding section of our fields planted in 2024 instead of only some samples per plot. The results from 110 plots (about 2,200 phenotyped plants) confirmed that plants positioned at the ends of the row tend to have more aerial root-containing nodes and roots per node and thicker stalks and aerial roots. We have conventionally called this phenomenon the "edge effect". We hypothesized that several environmental factors could be linked to this phenomenon, such as humidity gradient, competition for fertilizers, and light influence. After some preliminary tests, the humidity factor was ruled out. Therefore, we decided to explore light-related aspects this year. Goal 3: Evaluate the nitrogen benefits and potential yield trade-offs across multiple environments. During the early stages of our pre-breeding program, we had crossed three tropical maize landraces with three Midwest-adapted elite inbreds (all of them holding a recently expired Plant Variety Protection Act certificate) to develop adapted recombinant inbred lines with nitrogen fixation-related traits based on aerial root phenotypes. After being subjected to four generations of inbreeding, these lines showed varying counts of nodes with aerial roots. At the end of 2023, we had selected nine of these lines. We crossed them with a complementary tester (that is, a genotype also Midwest-adapted but chosen within a heterotic group different from that to which the elite parent of each line belongs). Then, in 2024, in a field trial conducted in Hancock, WI, the F1 hybrids obtained were grown in soils under severe nitrogen deficiency (-80 points of nitrogen per acre) and inoculated with diazotrophs. Just like their pre-breeding program-conceived parents, these hybrids also exhibited differing numbers of aerial root-containing nodes. However, the average number of nodes with aerial roots in the hybrids was proportionally lower than the average previously observed in those parents, denoting the additive effects of the genes contributing to this trait. The four hybrids with the largest number of aerial root-containing nodes showed higher chlorophyll content, yield, and grain weight values under very low nitrogen conditions. A very high correlation (≈ 0.85 calculated by Pearson's product-moment correlation coefficient method) was observed between yield and the number of aerial root-containing nodes. These results suggest that introgressing aerial root-related traits into Midwest-adapted maize inbreds is feasible and may reduce nitrogen fertilizer requirements without significant productivity losses.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Leveraging bacterial and plant genetics to optimize associative nitrogen fixation for cereal crops. Jean-Michel An�, Department of Genetics from the University of Wisconsin - Madison, Madison, Wisconsin, on October 9, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Biological nitrogen fixation on the aerial roots of maize for sustainable agriculture. Jean-Michel An�, Wisconsin Crop Improvement Association Annual Meeting, Middleton, Wisconsin, on December 3, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Biological nitrogen fixation on the aerial roots of maize and sorghum for sustainable agriculture. Jean-Michel An�, XXXII Plant & Animal Genome Conference 2025 (PAG32) on January 11, 2025
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Mining tropical maize for traits associated with aboveground symbiotic nitrogen fixation. Jason Wallace, XXXII Plant & Animal Genome Conference 2025 (PAG32) on January 11, 2025
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Engineering nitrogen-fixing bacteria associated with maize and sorghum roots for sustainable agriculture. Jean-Michel An�, XXXII Plant & Animal Genome Conference 2025 (PAG32) on January 12, 2025
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Gene-by-environment interactions between hosts and their microbiomes. Dr. Jason Wallace, XXXII Plant & Animal Genome Conference 2025 (PAG32) on January 14, 2025
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