Source: IOWA STATE UNIVERSITY submitted to NRP
IDENTIFICATION AND CHARACTERIZATION OF PARALLEL SPINDLE GENES TO RESTORE HAPLOID MALE FERTILITY IN MAIZE DH BREEDING TECHNOLOGY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030185
Grant No.
2023-67013-39443
Cumulative Award Amt.
$649,443.00
Proposal No.
2022-10295
Multistate No.
(N/A)
Project Start Date
Jul 1, 2023
Project End Date
Jun 30, 2026
Grant Year
2023
Program Code
[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production
Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
(N/A)
Non Technical Summary
Growing population and shrinking agricultural resources are one of the most important challenges facing food production. In order to meet the projected increase of global demand for food, feed, and fiber (100% by 2050), the linear progress will need to be increased by accelerating the efficiency, reliability, and speed of genetic improvement. Doubled haploid (DH) technology can speed up inbred line development by reducing the number of generations from seven in traditional breeding to two generations in DH breeding. There are two main steps in DH technology. The first step in a DH breeding program is to produce haploids for a donor genotype by pollinating the donor using a maternal haploid inducer as male. The second step is to produce DHs by self-pollinating the haploids. Haploid maize has a decent amount of haploid female fertility, but lack of haploid male fertility (HMF) is the bottleneck to develop DH lines efficiently. To overcome the HMF limitation, haploid seedlings are chemically treated with colchicine to mediate genome doubling, which is labor- and resource- intensive and it is an inefficient method. The alternative is to find genetic mechanism(s) to restore HMF.Our previous research in Arabidopsis informed us that mutations in parallel spindle (PS) genes are sufficient to restore HMF in Arabidopsisand the DHs obtained using ps mutations have no chromosomal abnormalities. The goal of this research is to identify maize PS genes with sequence similarity to Arabidopsis PS genes. Mutations by transposon insertion in maize PS genes will be identified from public collection and new mutation will be created by genome editing experiments. Haploids will be produced for these mutant plants and their male fertility will be evaluated by scoring for the presence of anthers and pollen. Ultimately, this project will identify genetic mechanism(s) to restore HMF without the use of labor- and resource- intensive and toxic chemical methods. In other words, haploid seeds can be directly sown in the field. Genetic mechanism(s) to restore HMF has the potential to make DH technology in maize broadly available, which is still mostly confined to major crop breeding programs. Further, these genes are conserved in other plant species opening the possibility for the application of this technology to other crops where it is not available so far.
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
2011510108070%
2011510103020%
2011510104010%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1080 - Genetics;
Goals / Objectives
Our previous research in Arabidopsis informed us that mutations in parallel spindle (PS) genes are sufficient to restore haploid male fertility in Arabidopsis. The major goal of this project is to identify and characterize homologous PS genes that can restore haploid male fertility (HMF) in maize haploids and to evaluate their applicability in various maize genetic backgrounds.The objectives of this study are to:(1) identify zmps and zmjason (collectively, ps) mutations that restore HMF in maize,(2) characterize the maize spontaneous doubled haploids resulting from ps mutations,(3) characterize the diploid maize ps mutants, and(4) conduct a pilot study to determine whether ps mutations can restore HMF in maize in different genetic backgrounds.
Project Methods
Efforts:Parallel spindle and Jason (collectively, PS) transposon insertion mutants will be isolated by marker assisted selection and backcrossing from UniformMu and BonnMu mutant collections.Plasmid DNA constructs with guide RNA for genome editing using CRISPR/Cas9 will be generated by standard cloning procedures.Transformable maize B104 will be used for genome editing by an Agrobacterium-mediated transformation protocol.Edited plants will be selected by T7 endonuclease assays followed by Sanger sequencing to validate the edits.Pollen and leaf DNA content will be estimated by ploidy analysis in a flow cytometer.Male meiocytes from anthers will be prepared on a microscopic slide and stained with DAPI to visualize chromosome segregation.Genotypic information of the doubled haploids (derived from ps mutant) across the maize genome will be acquired by using DArTseq genotyping technology.Acetocarmine staining will be used to prepare root tissues on microscope slides to visualize condensed chromosomes and counting (karyotyping).Grain yield or other agronomic parameters will be collected from field grown plantsHaploids will be produced in vivo by using haploid inducer as males and the donor (for which haploids to be produced) as females in cross-pollinationHaploid male fertility will be assessed by scoring for the presence of anthers and pollen as described in previously published protocolsEvaluation:Year 1:Milestone1: ps haploid mutants evaluated for haploid fertility relative to wild typeMilestone2: Pollen DNA content by ploidy analysis and female fertility of ps mutant haploids evaluatedMilestone3: Plasmid DNA construct made to edit PS genesMilestone4: B104 transformation with the constructs completedYear2:Milestone5: Edited plants identified and increasedMilestone6: Doubled haploids (derived from ps mutant) are producedMilestone7: Diploid ps mutant characterized by ploidy and karyotyping analysisMilestone8: Microscopy experiments of male meiosis in ps mutant haploid and diploid completedMilestone9: Yield and other agronomic traits of ps diploid mutant collectedYear3:Milestone10: Genotypic information of the doubled haploids (derived from ps mutant) across the maize genome acquired by DArTseq genotyping technologyMilestone11: Haploid male fertility evaluated for the new genome edited mutantsMilestone12: The applicability of ps mutations in various genetic backgrounds to restore HMF is evaluated.Indicators of success will be measured by publications, conference presentation, new germplasm and students trained. We expect at least 3 peer-reviewed publications and at least 3 conference presentations are completed at the end of the project. New mutant germplasm is developed and available for application and future research. 1 graduate student will have acquired training in plant breeding and molecular genetics research, which will be demonstrated by their ability to present in conference and/or peer-reviewed publication.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:Plant breeders Scientists Plant breeding and seed companies National and international breeding organizations Graduate and undergraduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provides training and professional development to a PhD student in Interdepartmental Genetics and Genomics at ISU. The student directly works with the project and gains hands-on experience with a range oflaboratory and field methods. In addition, regular meetings are held between the student and the PI to monitor progress and one-on-one mentoring. The student participated in a workshop to gain skills in microscopy techniques at Dr. James Birchler's lab at U of Missouri, Columbia, MO. The student also participated in a CRISPR workshop held at ISU to gain hands-on experience in genome editing methods. A research scientist also gained experience in learning genome editing techniques by involving in designing andbuilding plasmid vector constructs to edit ZmPS1 gene in maize. A visiting student from Thailand got exposed to genome editing through this project by involving in designing and building plasmid vector constructs to edit ZmJR genes in maize. An undergraduate student got involved in the project and earned skills in performing flow cytometry, microscopy and genotyping techniques. How have the results been disseminated to communities of interest?The results have been disseminated by poster presentations at international conferences and seminars at ISU. Posters presented Aboobucker SI, Zhou L, Frei UK, Lubberstedt T (2024) Parallel spindle genes restore haploid male fertility - removing a bottleneck in doubled haploid technology. 66th Annual Maize Genetics Meeting, Feb 29-Mar 3, Raleigh Convention Center, Raleigh, NC. Aboobucker SI, Zhou L, Frei UK, Lubberstedt T (2023) Parallel spindle genes restore haploid male fertility - removing a bottleneck in doubled haploid technology. American Society of Plant Biologists Meeting, Aug 5-9, Savannah Convention Center, Savannah, GA. Seminars Aboobucker SI(2024) Manipulating meiosis to restore haploid male fertility inplants. Lubberstedt Group Seminar, Jun 7, Iowa State University, Ames, IA. Aboobucker SI (2023) Manipulating meiosis to restore haploid male fertility in plants. Crop Bioengineering Center Symposium, Dec 7-8, Iowa State University, Ames, IA. Aboobucker SI (2023) Manipulating meiosis to restore haploid male fertility in Arabidopsis. Interdepartmental Plant Biology, Oct 4, Iowa State University, Ames, IA. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Growing population and shrinking agricultural resources are one of the most important challenges facing food production. In order to meet the projected increase in global demand for food, feed, and fiber (100% by 2050), the linear progress will need to be increased by accelerating the efficiency, reliability, and speed of genetic improvement. Doubled haploid (DH) technology can speed up inbred line development by reducing the number of generations from seven in traditional breeding to two generations in DH breeding. There are two main steps in DH technology. The first step in a DH breeding program is to produce haploids for a donor genotype by pollinating the donor using a maternal haploid inducer as male. The second step is to produce DHs by self-pollinating the haploids. Haploid maize has a decent amount of haploid female fertility, but lack of haploid male fertility (HMF) is the bottleneck to develop DH lines efficiently. To overcome the HMF limitation, haploid seedlings are chemically treated with colchicine to mediate genome doubling, which is labor- and resource- intensive and it is an inefficient method. The alternative is to find genetic mechanism(s) to restore HMF. Identifyzmpsandzmjason(collectively,ps) mutations that restore HMF in maize, 1.1. Transposon insertion maize mutants of parallel spindle (ZmPS1) and JAS-Related (ZmJR1 to ZmJR4) genes isolated from genetic stocks through genotyping and Sanger sequencing. 1.1.a. Haploids were produced for Zmps1 mutant and we found tha the mutant restored haploid male fertility in maize.Haploid male and female fertility data collected from Zmps1 mutant and controls. Pictures of the mutant haploid and respective controls were obtained along with flow cytometry histograms to analyze ploidy of the mutant and control plants. 1.1.b. Haploids were produced for Zmjr1 to Zmjr4 mutants. These haploids will be evaluated for its ability to restore haploid male fertility in Summer 2024 in Iowa. 1.2. Plasmid vectors to edit the ZmPS1 or ZmJR genes have been constructed and maize inbred oine B104 will be transformed with these constructs at the Crop Bioengineering Lab, ISU. Objective 2:characterize the maize spontaneous doubled haploids resulting frompsmutations 2.1. Zmps1 maize mutant is crossed to W22, a polymorphic genetic background to the mutant and haploids were produced in Winter 2023. The haploids are grown in Summer 2024 to produce DH lines. Genotyping data will be collected from the haploids and the resulting DH lines to confirm the absence of any abnormality by the ps mutation. Objective 3:characterize the diploid maizepsmutants 3.1 Molecular characterization of the diploid Zmps1 mutant is complete. The TE element inserted in the mutant is sequenced and gene expression data obtained for the mutant and the respective control. 3.2. Microscopic slides are being produced to analyze the male meoicyes of the Zmps1 and Zmjr mutants. 3.3. Zmps1 mutation is currently being crossed with W22 and Mo17 in Summer 2024to study the effect of any potential pleoitropic effects. Per se and hybrid performance will be evaluated in Summer 2025. 3.3. Similar experiments are underway for the Zmjr mutants. Objective 4:conduct a pilot study to determine whether ps mutations can restore HMF in maize in different genetic backgrounds. Zmps1 mutant with high haploid male fertility is being introgressed into several other backgrounds to evaluate the effect of mutation in restoring haploid male fertility in different backgrounds. Currently, BC1 lines are produced and BC2 will be obtained in Summer 2024, followed by haploid induction in Winter 2024. The haploids will be evaluated in Summer 2025.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Aboobucker SI, Zhou L, L�bberstedt T (2023) Haploid male fertility is restored by parallel spindle genes in Arabidopsis thaliana. Nature Plants 9: 1-5 https://doi.org/10.1038/s41477-022-01332-6
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Aboobucker SI, L�bberstedt T (2023) A genetic mechanism to restore haploid male fertility in Arabidopsis  an alternative to chemical methods. Nature Plants. https://doi.org/10.1038/s41477-022-01335-3