DISCOVERY AND CHARACTERIZATION OF NOVEL GENES REGULATING PLANT ARCHITECTURE IN MAIZE USING FIRST FILIAL HYBRID ASSOCIATION MAPPING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028244
• Grant No.: 2022-67012-36601
• Cumulative Award Amt.: $224,752.00
• Proposal No.: 2021-08381
• Project Start Date: Jan 1, 2022
• Project End Date: Jun 30, 2025
• Grant Year: 2022
• Program Code: [A1152]- Physiology of Agricultural Plants

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Biochemistry

Non Technical Summary
Plant shape or architectureis critical toincreaseproductivity,optimize inputs, andachieve sustainable solutionsto meet national demands for human and animal nutrition. Ideal plant architecture safeguards against losses due to abiotic stresses such as lodging and optimizes yield per acre via light interception and plant density. The proposed research targets two important components of plant architecture, plant height and leaf angle, that arecontributors to yield maximization in maize.The proposed research will identify novel genes that can modify these traits.The findings of this study will be published in peerreviewed and open-access journals. The data generated from all the experiments will be made available to thepublic through data repositories.The molecular identification of genes controlling plant height and leaf angle will help in their detection in other mapping frameworks and could be used bymaize breeders to develop improved maize germplasm.

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
201	1510	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics;

Keywords

bulked segregant analysis and natural variation

genomics

quantitative genetics

transcriptomics

Goals / Objectives
The objective of this proposal is to utilize a genetic screen using dominant maize mutants that show variation in leaf angle (LA) and plant height (PH) to identify novel loci and alleles regulating plant architecture.My long-term goalis to develop a cost-effective gene discovery platform in maize utilizing existing genomic resources to identify novel genes regulating plant architecture.To meet these goals, I have proposed anF1 hybrid association mapping (FOAM) approach that uses dominant architectural mutants of maize in crosses to a panel of diverse maize inbred lines with known SNP genotypes. I will quantify trait severity of the mutant phenotypes in hundreds of F1 hybrid progenies, and conduct GWA to identify loci at high genetic resolution that modify mutant phenotype. The discovery of novel loci will not only improve our basic understanding of plant morphology but has the potential to improve maize yield. I propose the following objectives:Perform FOAM using dominant mutants of maize influencing PH and LA to discover genes modifying these traitsIdentify causative genes for novel mutants of PH and LAConfirm molecular identity of candidate genes by generating independent allelesAnalyze gene expression consequences in dominant mutants of PH and LA

Project Methods
This proposal aims atidentifying and characterizing novel loci and allelic diversity of plant architecture in maize using mutant reporters impacted in plant height (PH) and leaf angle (LA). To achieve this goal, the proposal has four research objectives. The methods used to achieve these objectives are outline below.Objective 1.Discover genes modifying PH and LA using FOAM with dominant mutants: I will use two known GA signaling mutants (D8andD9), three uncharacterized dominant dwarfs (D13,D2516, andSemi-Dwarf2), and three LA mutants (Lg3,Lg4, andLg*-NG) to develop F1 hybrid populations with the maize association panel that comprises 382 maize inbred lines. I will cross all dwarf mutants andLg*-NGwith the association panel in summer of 2021. TheLg3andLg4mutants will be crossed with the association panel during the winter season nursery. The F1 hybrid progenies will be evaluated as a single row plots with two replications in summer of 2022 at the Purdue University research farm. Each F1 hybrid progeny in the F1 association mapping (FOAM) population will segregate ~1:1 for the wildtype and mutant siblings. All F1 populations will be phenotyped at three weeks after planting, six weeks after planting, and after flowering. PH data will be collected as the distance of the top leaf collar from the soil. LA will be measured on the top collared leaf at three and sixweeks and at the leaves above the primary ear and below the flag leaf at maturity. Barcoded measuring devices and handheld barcode scanner will be used for high throughput data acquisition. Additional phenotypes, including total leaf number, days to anthesis, and stalk width will be collected to explore pleiotropy. The phenotypic data from mutants, wildtypes, and their ratio will be used to calculate least square means usinglsmeans.I have generated a computational workflow to perform genome-wide association (GWA) using this panel. The least square means will be used for GWA mapping using the compressed mixed linear model. Gene expression variation is a major driver for genetic variability in maize. I will also obtain public gene expression data set derived from a maize association panel. The transcript abundance of candidate genes from the FOAM experiments will be used for expression-GWA to identify cis-eQTL and test concordance with the trait GWA results.Objective 2. Identify causative genes for novel mutants affecting PH and LA: The molecular identities of four out of eight mutant tester alleles are published (D8, D9, Lg3,andLg4) but is unknown forD13,D2516,Semi-Dwarf2, andLg*-NG. The mutant alleles with uncharacterized polymorphisms were isolated from non-B73 backgrounds and later introgressed into B73. For each mutant, F1 heterozygous plants crossed to B73 will be self-pollinated to generate F2 populations. All mutants show a severe phenotype in homozygous condition. DNA from a single homozygous individual will be sequenced and reads will be aligned to the B73 RefGenV4 assembly using thebwaalignerand SNPs will be called usingSAMtools.The non-B73 introgressed segments containing the mutant locus will be identified by the high density of SNPs contributed by the background-of-origin for each mutant. To fine map the causative mutations, DNA will be extractedfrom a pooled sample of 200 homozygous mutants from the F2 progenies described above and sequenced. The allele frequencies will be calculated at each SNP. A sum of the allele frequency over a sliding window will be plotted and the region without B73 alleles will indicate position of the mutant loci. Because EMS induces G->A or C->T changes, and all four of the novel mutant alleles were derived from populations mutagenized with EMS, SNP genotypes in each mutant bulk will be inspected for homozygous G->A or C->T changes in the protein-coding sequence of the genes in the mapped interval.Objective 3.Confirm the molecular identity of dominant mutants: The candidate genes identified in Objectives 1 and 2, for the four uncharacterized mutant alleles, will be validated by screening for additional alleles. In year 2 of this project, I will obtain coding sequence insertion alleles in the high-confidence candidate genes identified in Objective 2 and cross them with the dominant mutants. As all four mutant alleles are semi-dominant, if I knock out the wildtype allele in these mutants I predict it will enhance the mutant F1 phenotype and I should observe severe mutant plants segregating in the progenies from these crosses. Alternatively, I will generate loss-of-function alleles using pollen EMS mutagenesis, that generates new alleles at a frequency of ~1/1000 plants. For this approach, pollen from wildtype B73 will be treated with EMS and used to pollinate ears of heterozygous dominant mutants. 500 ears of each mutant heterozygote will be pollinated, yielding ~15000 M1 seeds for each mutant. In the M1 progenies, I expect to recover approximately seven severe mutant plants for each mutant. Severe mutant plants will be propagated by outcrossing to wildtype B73 plants. Leaf tissue will be collected from severe mutants and PCR amplification and sequencing will be used to test for mutations within the candidate gene(s) from Objective 2.Objective 4. Analyze gene expression consequences in dominant mutants of PH and LA: In addition to characterizing the causative genes underlying plant architecture mutants, I will identifypathways perturbed in these mutants. The uncharacterized mutants in B73 background segregating ~1:1 for heterozygous mutant and wildtype siblings will be used for RNA sequencing (RNAseq). For the PH mutants (D13,D2516,Semi-Dwarf2), immature shoot apices including neighboring stem tissue will be dissected three weeks after planting.For theLg*-NGmutant, leaf collar of the top collared leaf will be harvested from mutant and wildtype siblings at three weeks. Three biological replicates from fifteen plants each will be obtained for each mutant and wildtype sample. Paired-end reads from these sampleswill be aligned to the B73 RefGenV4 genome to obtain a list of differentially expressed genes (DEG). DEG will be used for pathway-level analyses using KEGG pathways. These genes will be compared to maize gene expression datasets available in the Dilkes lab that includes BR, GA, and auxin treatments and mutants.Evaluation Plan Evaluation of research: Success in year 1 will be indicated by completion of GWA on six out of the eight FOAM populations. Candidate gene lists and validation should commence at the end of the first year. Largely independent objectives in this proposal will allow sequencing for both mutant identification (objective 2) and transcriptome analyses (objective 4) in the first year. I will regularly meet with my mentor to provide updates on the progress of the project and discuss research findings. During these meetings, we will discuss the prioritization of SNP-trait candidates and draft the first manuscript by the beginning of the second year of the project. My mentorship team will also provide research evaluation at our biannual meetings.Evaluation of career development: Periodic evaluation of my individual development planwith my mentor will help track my research and professional accomplishments. The mock interview for academic jobs will provide me with evaluation of my interpersonal skills and identify areas where I could improve. I will work with my mentor on development of course curriculum and give guest lectures in the course taught by him. I will get anonymous feedback from students and my mentor at the end of these lectures to evaluate my teaching skills. This will help me improve lecture contents and delivery. Feedback on research presentations in lab meetings, departmental seminars, and conferences will provide independent assessment of presentation development and communication skills. My mentorship team will also provide independent assessment of career development at our biannual meetings.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience: As stated in the previous report, thisproject is research-based and does not contain any extension objectives. In the past year,I gave a research talk in the Plant Phenomics Phridays Seminar organized by Dr. Carolyn Lawrence-Dill at theIowa State University. I also gave an invited talk at the 5th Canadian Wheat Symposium at the University of British Columbia, Canada. The participants at both these gatheringsincluded undergraduate and graduate students, postdoctoral researchers, early career scientists, and faculty from public universities in the US and Canada, and scientists from the Ag industry. I also presented posters on my research at the Annual Maize Genetics Meetings held in St. Louis, MO. I have published two manuscriptin the last year, one in G3 and the other in the Plant Journal,where the funding from this fellowship was acknowledged. Changes/Problems:I am using alternate strategies to isolate alternate suppressor or enhancer alleles for Lg*-NG mutant allele that impacts leaf angle of maize. This mutant has a very strong phenotype in the Mo17 background.The phenotype of the homozygotes and heterozygotes in Mo17 background is hard to distinguish. This mutant is strongly suppressed by B73 background. Last summer, I have started backcrossing this mutant into B73 to carry out the targeted EMS mutagenesis using pollen from a homozygote in a suppressing genetic background. What opportunities for training and professional development has the project provided?Last yearI was invited for a talk to the 5th Canadian Wheat Symposium at the University of British Columbia, Canada. At this symposium, I interacted with wheat researchers, primarily geneticists and breeders from Canada and the US. At this meeting, I embarkedon new collaborations with wheat breeders in the two countries to develop methodologies originally developed through this fellowshipto validate genes for disease resistance in wheat, especially leaf rustand stripe rust. At this meeting, I also learned about new job opportunities in wheat pathology that would allow me to carry similar research in systems other than maize. Altogether, I got an opportunity to engage with wheat community that I would have missed otherwise. I worked with my fellowship committee that comprisesof Drs. Brian Dilkes, Damon Lisch, Erin Sparks, and Peter Balint-Kurti to prepare academic applications. I applied for 20 assistant professor positions this past year and got an interview with the Department of Biochemistry at the Clemson University. I am currently waiting for the results from the hiring committee at Clemson for the next round of interviews. How have the results been disseminated to communities of interest?Multiple efforts have been made, some intentional and other pure coincidence, to disseminate the work from this award. I have published two research articles that employed techniques to carry genetic experiments described in this proposal, with a third manuscript under review. I have also used these techniques in other crops especially sorghum to develop alternate alleles that have not been possible before. To my knowledge this would be the first demonstration of germinal allele recovery in sorghum through targeted EMS mutagenesis, an approach that would increase the pace of scientific research by providing a simple, yet effective tool to sorghum geneticists and breeders. I am extending this approach to wheat through new collaborations with wheat pathologists and breeders in the US and Canada. I have also given two talks in the last year where I discussed the work from this project and provided a research framework to study semi-dominant mutants and natural variation in maize. What do you plan to do during the next reporting period to accomplish the goals?By the next reporting period, I plan to publish three manuscripts that would be a direct outcome of this project. One of the manuscripts will be submitted this month, with two others to follow. I will continue to provideguest lectures and honemy teaching skillsthrough lectures in the undergraduate course that I have teaching for the last two years under the guidance ofDr. Erin Sparks at the University of Delaware. I will continue to work with my mentors Dr. Damon Lisch and Dr. Brian Dilkes to prepare my application packets for academic jobs.

Impacts
What was accomplished under these goals? D13right now. This will be followed by descriptionsof FOAM results fromD8,D9,Sdw2,D2516, andLg*-NGmutant alleles. For theD13mutant, FOAM identified a cis-acting polymorphism at the causative gene forD13to be the major modifier of plant height in the maize association panel. D13has already been identified. That work will be described in a manuscript that is currently being drafted. The candidate genes forSdw2,D2516, andLg*-NGhave also been identified and the molecular validation of these genes is underway as described in the next goal. Sdw2, and one suppressor ofSdw2-1was finished last year. I identified multiple mutations in one of the candidate genes forSdw2-1. The phenotypic analysis of these suppressors and enhancers was finished past summer and phenotypic results are consistent with these alleles as legitimate variants impacting theSdw2locus. Similar analysis hasbeen finished withD2516allele as wellwhere fiveenhancer alleles were identified and one of the candidate gene revealed multiple independent variants confirming the identity of the gene underlyingD2516mutant. TheLg*-NGallele has turned out to be more complicated due to lack of recovery of variants through multiple EMS genetic screens attempted this past summer. We are using alternate methods to recover suppressors or enhancers ofLg*-NGand the experiments for this approach are underway. Sdw2andD2516showed a remarkably similar gene expression. Surprisingly, both of these mutants identified genes that are also differentially expressed in other mutants in the lab including some implicated in immunity.Lg*-NGmutant also showed a very distinct expression profile that is very different from other mutants impacting leaf angle. I am using these transcriptomic datasets to develop hypothesis to understand the physiology and molecular pathways that lead to changes in plant architecture.?

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: M�line, V., Caldwell, D.L., Kim, B.-S., Khangura, R.S., Baireddy, S., Yang, C., Sparks, E.E., Dilkes, B., Delp, E.J. and Iyer-Pascuzzi, A.S. (2023), Image-based assessment of plant disease progression identifies new genetic loci for resistance to Ralstonia solanacearum in tomato. Plant J, 113: 887-903. https://doi.org/10.1111/tpj.16101
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Matt Sauer, Jianfei Zhao, Meeyeon Park, Rajdeep S Khangura, Brian P Dilkes, R Scott Poethig, Identification of the Teopod1, Teopod2, and Early Phase Change genes in maize, G3 Genes|Genomes|Genetics, Volume 13, Issue 10, October 2023, jkad179, https://doi.org/10.1093/g3journal/jkad179
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: The Auxin Response Factor ARF27 is required for maize root morphogenesis Linkan Dash, Maxwell R. McReynolds, Melissa A. Draves, Rajdeep S. Khangura, Rebekah L. Muench, Jasper Khor, Jodi B. Callwood, Craig L. Cowling, Ludvin Mejia, Michelle G. Lang, Brian P. Dilkes, Justin W. Walley, Dior R. Kelley bioRxiv 2023.08.15.553439; doi: https://doi.org/10.1101/2023.08.15.553439

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:This projectisprimarilyresearch-basedanddoesnotformallyhaveanyextensioncomponent.To disseminate the research findings of this project, I gave multiple research talks and guest seminars over the last year.Igave a research talk at the 64thAnnual Maize Genetics Meeting in St. Louis on March 31st, 2022. The participants of this meeting included undergraduate and graduate students, postdoctoral researchers, early career scientists, and faculty from public universities in the US, scientists and representatives from major seed and Ag-tech companies such as Bayer, Corteva, Syngenta, BASF, and INARI.I gave an invited research talk at the University of Delaware (Hosted by Dr. Erin Sparks) on March 1st, 2022. I gave another invited research talk in the Department of Biochemistry at North Carolina State University on December 1st, 2022 (Hosted by Dr. RubénRellan-Alvarez). The invited research talks were catered to undergraduate and graduate students, postdoctoral researchers, and faculty of STEM disciplines at these institutes. Besides research talks, I presented posters on my current research at two consecutive Annual Mize Genetics Meetings held in the years 2022 and 2023. I have one publication that is under review at Plant Physiology where the support of this fellowship award was acknowledged.? Changes/Problems:I did not have any major problems with my project so far. In the proposal, I identified a pitfall of F1 association mapping where the low frequency of modifiers for some maize mutants may not permit genome-wide association mapping to reveal associations. I developed a bi-parental backcross population for two such mutants and used bulk-segregant analysis to identify modifiers. What opportunities for training and professional development has the project provided?This project provided financial support for me to attend two Annual Maize meetings in 2022 and 2023. I presented my research through a research talk and poster sessions at these meetings. At these meetings, I networked with industry and academic leaders and initiated new collaborations. I gave two invited research talks through my collaborators which highlighted my research work and provided me with more venues for scientific communication. As highlighted in other sections on achievements, I gave guest lectures over two semesters in an undergraduate course at the University of Delaware that greatly increased my confidence as an educator and gave me the opportunity to work alongside my co-mentor Dr. Erin Sparks to learn best practices of teaching. How have the results been disseminated to communities of interest?As part of my training to become an educator, I gave in-person guest lectures in the 2022 and 2023 spring semesters in an undergraduate course PLSC 270/APEC 270 (Biotechnology: Science and Socioeconomic Issues) taught by my collaborator and co-mentor Dr. Erin Sparks at the University of Delaware. I used my research work supported by this fellowship award for class examples to cover topics such as the relationship between genotype and phenotype, genotype x environment interactions, linkage, and recombination. The cohort in this course comprised students who were broadly interested in agriculture. The class discussions of genetics, its importance, and methodologies using simple examples showcased overwhelming interest from multiple students that made these lectures really enjoyable as a teacher. Dr. Sparks later communicated to me privately that students really enjoyed these lectures and would like me to continue these lectures every year for this course at the University of Delaware. What do you plan to do during the next reporting period to accomplish the goals?I plan on publishing at least two scientific manuscripts by the next reporting cycle which will be a direct outcome of this project. The first manuscript will describe the maize mutant that provides resistance to multiple maize pathogens. The second manuscript will discuss the novel skim sequencing approach that utilizes next-generation sequencing to map maize mutants, and their modifiers, identify contaminants, and perform haplotype analysis using mutants of unknown pedigrees. Both of these manuscripts are under preparation as of now.I also plan on finishing the molecular characterization of the novel PH mutantSdw2for which I have already generated two suppressor alleles that will permit independent molecular validation. This work will also include transcriptomic analysis of the mutant as it will help in generating mechanistic insights into the molecular mechanisms involved in the modulation of plant height inSdw2.

Impacts
What was accomplished under these goals? The USDA NIFA Fellowship award provided financial and professional support for me to excel as a maize geneticist. I have identified the molecular basis of one maize mutant through the support of this award. This mutant provides resistance to multiple maize pathogens and is likely useful in maize breeding to create improved hybrids with broad-spectrum disease resistance. Based on this research work, I am currently drafting an IP through the Purdue Research Foundation at Purdue University. I am also in the process of molecular characterization of two short-stature maize mutants. The genomic localization experiments have identified regions of the maize genomes that have not been previously associated with any known gene in maize that regulates plant height. I have carried out independent experiments to validate these findings. The molecular identity of these potentially novel genes will provide additional resources to breeders to alter plant architecture to safeguard maize crops from damaging storms that are becoming a major challenge for corn production in the US corn belt. The project has the following four objectives and the major activities for each objective are provided below. 1. PerformF1 Association Mapping (FOAM)usingdominantmutantsofmaizeinfluencingplant height (PH)andleaf angle (LA)todiscovergenesmodifyingthesetraits:I have already developed and phenotyped F1 association mapping populations that were proposed under the scope of this project. This includes the population with multiple plant architecture mutants:Dwarf8(D8),D9,D13,D2516, andSemi-Dwarf2(Sdw2), and one novel leaf angle mutantLiguleless*-NG(Lg*-NG). As expected, the mutant phenotype was impacted to varying degrees by different F1 hybrid genetic backgrounds. I have also carried out genome-wide association analysis using this data and have identified putative QTLs that interact with these mutants. Furthermore, I have developed bi-parental populations using select F1 hybrids to perform bulk-segregant analysis to identify additional loci that modify these mutants. In the process of mapping these mutants and their modifiers, I have developed a new skim sequencing mapping approach that permits genetic mapping at $21 per sample. This approach has the potential to simplify genetic mapping and expedite molecular marker developments for marker-assisted selection and background selection in plant breeding. I am currently curating these modifiers that impact each of these PH and LA mutants to pave the way to identify their molecular basis. 2. IdentifycausativegenesfornovelmutantsofPHandLA:We have already identified the causative gene underlying the novelD13maize mutant but this work has not been published. For the other three novel maize mutantsD2516,Sdw2, andLg*-NGthat are in the scope of this project, I have identified a narrow genomic interval through a series of linkage mapping experiments. Additional experiments to further narrow down the genomic intervals and independent experiments to create additional alleles described below will help identify the candidate genes underlying these novel maize mutants. 3. Confirmthemolecularidentityofcandidategenesbygeneratingindependentalleles:In the summer of 2022, I identified two novel knock-out suppressor alleles ofSdw2using induced mutagenesis. These knock-out alleles ofSdw2have already been sequenced and the experiments to identify the causative polymorphisms underlying these suppressors ofSdw2are currently underway. This summer I will carry out similar mutagenesis experiments to create knock-out alleles ofD2516andLg*-NGto aid in the molecular characterization of the causative polymorphism(s) and the gene identities. I have developed a novel skim sequencing method based on Next-Generation sequencing that only costs $21 and can identify true suppressors or enhancers from a pollen contaminant and greatly enhances the efficiency of gene discovery. 4. AnalyzegeneexpressionconsequencesindominantmutantsofPHandLA:In the winter of 2022-23, I carried out transcriptomic experiments on theSdw2andD2516mutants in the greenhouse. I collected tissue from elongating internodes below the tassel from plants at V7 vegetative stage in the greenhouse. Tissue from 10-12 independent plants was pooled to make one biological replication for each wild-type and mutant sample. A total of three biological replications each for mutant and wild-type siblings were subjected to RNAseq. I am currently analyzing the transcriptome data from these mutants. I will compare the transcriptome data from these PH mutants to other PH mutants whose transcriptome data is either publicly available or has been generated internally in the Dilkes Laboratory to examine the gene expression consequences. This will help generateadditional hypotheses about the genetic mechanisms that might be at play in these novel PH mutants. I will carry out the transcriptomic experiment onLg*-NGin an outdoor grow this summer.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Khangura R. S., G. S. Johal, and B. P. Dilkes, 2022 Genome-wide association identifies impacts of chlorophyll levels on reproductive maturity and architecture in maize. bioRxiv. https://doi.org/10.1101/2022.11.07.515492. In Review at Plant Physiology