Source: SOUTH DAKOTA STATE UNIVERSITY submitted to NRP
EVALUATION OF NATURAL AND GENOME-EDITED GENETIC VARIATIONS IN AUXIN AND CYTOKININ PATHWAY GENES FOR OPTIMAL NITROGEN FIXATION IN SOYBEAN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032176
Grant No.
2024-67014-42322
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-08568
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
(N/A)
Non Technical Summary
Biological nitrogen fixation in root nodules of soybean helps reduce the need for expensive, energy-intensive, and environmentally damaging synthetic nitrogen fertilizers. Nodule nitrogen supply peaks during early reproductive stages (R1-R3) and declines thereafter, whereas grain nitrogen demand increases in the later reproductive stages (R5-R7). Therefore, high-yielding soybean varieties take up nitrogen from the soil, depleting soil nitrogen reserves that would otherwise be available for the future crops necessitating research to increase nodule nitrogen supply in later reproductive stages. We hypothesize that soybean genotypes with a higher number of nodules with delayed maturation can continue to fix nitrogen at these reproductive stages and help meet nitrogen needs in a sustainable manner. Prior results and preliminary data from the PD's laboratory as well as those from other research suggest that altering the balance between two plant hormones auxin and cytokinin can lead to higher nodule numbers with delayed maturity. This research project will (1) evaluate genome sequences of 1,110 accessions from the USDA soybean germplasm collection to identify natural genetic variants in genes associated with auxin and cytokinin biosynthesis, (2) validate their roles thorough genome editing and laboratory analysis, and (3) characterize soybean germplasm with specific genetic modification for optimal nitrogen fixation. The accessions or derivatives including knowledge would help develop soybean varieties with optimal nitrogen fixation that do not deplete soil nitrogen or required additional nitrogen fertilizer application.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011820102030%
2061820102070%
Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1020 - Physiology;
Goals / Objectives
Biological nitrogen fixation provides only 50 to 60% of soybean's nitrogen needs. Nodule nitrogen supply declines during later reproductive stages R5 and R7 presumably due to nodule senescence. This leads to depletion of soil nitrogen by soybeans or in some cases necessitates application of additional nitrogen by soybean producers at these stages. We hypothesize that soybean genotypes with a higher number of nodules with delayed maturation can continue to fix nitrogen at these reproductive stages and help meet nitrogen needs in a sustainable manner. Prior findings suggest that reducing auxin output and/or increasing cytokinin output can lead to higher nodule numbers. The proposed project is expected to identify auxin and cytokinin genes responsible for nodule numbers, delayed nodule maturity and senescence in soybean.Optimizing nodule number, maturation and senescence provides the nitrogen fixed by the nodule available to the plant during the reproductive stages of soybean development, thereby helping sustainably meet soybean nitrogen needs.The objectives in this project are:1. To identify natural genotypic variation in auxin and cytokinin biology genes using 1000 soybean genomes data.2. Generating and characterizing CRISPR edits in hairy roots targeting components of auxin and cytokinin biology genes.3. Phenotyping selected natural genetic variants and genome-edited lines for hormone responses, nodulation, and nitrogen fixation.
Project Methods
For Objective 1, SnpEff tool (abbreviation for SNP effect) annotates the identified variants as "modifier" (representing mutations found in intergenic regions) "low" effect (leading to synonymous mutations), "moderate" effect (resulting in non-disruptive alterations in the protein structure) and "high" effect (causing disruptive changes to protein), considering the kind and placement of genetic variation. In Objective 2, Genome editing will be performed for corresponding genotypes or lines with "high level" genetic variations. Edited hairy roots will be tested for hormone responses and nodulation parameters nodule numbers and nitrogen fixation capacity and compared to "unedited" control roots using statistical analysis to determine significant changes if any. For Objective 3, Selected genotypes/lines with "high level" genetic variations from SnpEff analysis will be tested for nodulation efficiency using nodule number, nodule maturation times, nitrogen fixing capacity during reproductive stages. Statistical analysis will be used to determine significant changes in these parameters to evaluate the effect of genetic variation on nodule development and nitrogen fixation.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Plant Science and microbiology researchers including faculty, post-docs, students, and other research staff. Results are yet to be disseminated in scientific conferences. Changes/Problems:Since the soybean accessions maturity groups varied from 000 to X, there were some challenges in amplifying enough seeds to conduct follow-up characterization experiments. Another round of greenhouse growth would be required for obtain enough seeds. Initial PCRs for amplifying the genomic regions carrying SNPs did not work for some accessions likely due to the presence of PCR inhibitor pigments. DNA isolation and PCR conditions need to be optimized for specificity and target amplification by designing new set of primers and using different protocols for genomic DNA isolation. We will request a six month no cost extension due to the unanticipated delays. What opportunities for training and professional development has the project provided?Bhanu Petla, postdoc in this project has gained experience in procuring and maintaining soybean accessions. He also gained experience in designing and generating large number of CRISPR constructs. An undergraduate student Shishir Roka and a grad student Gurdeep Singh were trained on various molecular biology techniques including PCR, electrophoresis, CRISPR guide RNA design, Goldengate and gateway cloning, growing soybean plants and performing hairy root transformation. How have the results been disseminated to communities of interest?Bhanu Petla will be presenting a poster of the current work progress at the ASBP Plant Biology 2025 meeting, Milwaukee, WI from July 25-30, 2025. What do you plan to do during the next reporting period to accomplish the goals?Goal One: To identify natural genotypic variation in auxin and cytokinin biology genes using 1000 soybean genomes data. Genotypes identified in the analysis will be requested from GRIN network for multiplication as well as SNP verification by sequencing. Goal Two: Generating and characterizing CRISPR edits in hairy roots targeting components of auxin and cytokinin biology genes. Constructs targeting all the auxin and cytokinin pathway genes will be generated, sequenced and transformed into Agrobacterium rhizogenes. These constructs will be used for transforming soybean to generate transgenic hairy roots. Edits for these genes will be confirmed by PCR and sequencing. Edited roots will be used for further characterization of the constructs including nodule numbers and nitrogen fixation ability. Goal Three: Phenotyping selected natural genetic variants and genome-edited lines for hormone responses, nodulation, and nitrogen fixation. Selected soybean genotypes and CRISPR-modified lines will be grown in greenhouse or growth chamber and will be further evaluated. Theses lines will be tested for nodulation using USDA 110 Bradyrhizobium strain, hormone responses and nitrogen fixation during vegetative and reproductive stages.

Impacts
What was accomplished under these goals? Goal One: To identify natural genotypic variation in auxin and cytokinin biology genes using 1000 soybean genomes data. 100% Accomplished. Analysis of 1000 genomes data for identifying natural genotypic variation in auxin and cytokinin biology genes was carried out using SnpEff program (Cingolani et al. 2012). Soybean Lee pangenome data (Bayer et al. 2022) with 1110 soybean accessions was retrieved from Research repository of University of Western Australia. Expanded list of auxin and cytokinin pathway genes with their gene intervals was generated for Lee v1.1 genome. SnpEff analysis was carried out on South Dakota State University high performance computing cluster. SnpEff analysis identified a total of 698 SNPs classified as moderate effect or non-synonymous with amino acid change (37), low effect or synonymous with no amino acid change (68) and modifier with changes in non-coding regions (593). Analysis with the expanded list of auxin and cytokinin pathway genes and 1110 soybean genomes did not detect any high level SNPs that would disrupt the gene function. Overall, SNPs were identified in 19 auxin biosynthesis/signaling pathway genes and 18 cytokinin biosynthesis/signaling genes. Impact statement: SnpEff analysis of auxin and cytokinin pathway genes across pangenome identified new medium and low effect SNPs. Goal Two: Generating and characterizing CRISPR edits in hairy roots targeting components of auxin and cytokinin biology genes. 30% Accomplished. Genomic sequences for six auxin pathway genes (3-YUCCA and 3-GH3, showing moderate level mutations from the preliminary data) proposed were retrieved from Phytozome database. Two guide RNAs were designed targeting each gene using online guideRNA design tool chop-chop (Labun et al. 2019). Six constructs for 3-YUCCA and 3-GH3 auxin pathway genes were generated, sequenced and transformed in Agrobacterium rhizogenes. These constructs were used for generating transgenic hairy roots in wildtype soybean Williams 82. Impact statement: Six CRISPR constructs were generated targeting YUCCAA and GH3 and were transformed in Agrobacterium rhizogenes and hairy roots were generated. These lines will be tested for editing and characterized for nodulation phenotypes. Goal Three: Phenotyping selected natural genetic variants and genome-edited lines for hormone responses, nodulation, and nitrogen fixation. 25% Accomplished. Selected soybean genotypes (30) listed in the proposal from the initial analysis with SNPs in auxin pathway genes YUCCA and GH3 were procured from GRIN database and were grown in green house. Accessions represented both species of Glycine soja and Glycine max with light seed coat as well as pigmented dark seed coat. The accessions also represented various mature groups ranging from 000 to X. A single step genomic DNA isolation protocol with alkali PEG (Chomczynski and Rymaszewski 2006) was used to isolate gDNA from leaf samples. PCRs were carried out using gene specific primers to amplify regions containing the polymorphisms. SNPs were verified by PCR and sequencing for seven genotypes so far and analysis for the other genotypes is underway. Soybean genotypes and CRISPR lines will be tested for nodulation and hormone responses. Impact statement: Initial sequencing results confirm the SNPs reported in pangenome for seven genotypes. This validates the pangenome data at individual SNP level for its accuracy.

Publications