Progress 06/01/23 to 05/31/24
Outputs
Target Audience: The main target audience for this work is undergraduate students, graduate students, postdoctoral scientists, industry scientists, and faculty members. During this first year of the project, we presented a research seminar at the University of Wisconsin-Madison campus that provided an update on our work towards creating haploid inducers for carrot breeding. This seminar was attended by undergraduate students, graduate students, postdoctoral scientists and faculty members interested in plant biology, plant breeding, and plant cell and molecular biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The experimental work for this project is being carried out by a post-doctoral scientist who was hired for this project. The project provides them with the opportunity to gain experience carrying out gene editing in carrot and testing haploid induction. The project also provides them with the opportunity to present their work through seminar presentations and at conferences. How have the results been disseminated to communities of interest? We have published a manuscript describing our initial observation of haploid induction caused by mutation of carrot CENH3. This was published in Frontiers in Plant Science. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue to produce additional gene edited carrot plants carrying novel mutations in the CENH3 gene to test the haploid induction efficiency of these novel alleles. As novel mutant lines are produced, they will be put into a genetic crossing pipeline in which each mutant line will be crossed with a wild-type crossing partner. Progeny from each CENH3 mutant line will then be screened by PCR-based genotyping to identify candidate plants for further testing to determine if they are haploids. Using this approach, we will document the haploid induction potential of a number of new CENH3 mutant lines. In addition, we will also initiate experiments to test the effect that growth temperature has on the haploid induction efficiency of carrot CENH3 mutant lines. This work will be based on the precedent established in Arabidopsis thaliana, where elevated temperature has been shown to dramatically increase the rate of haploid induction. For these experiments, we will perform genetic crosses between the CENH3 mutant line described in our first publication and wild-type carrots growing at either 21°C or 29°C. Progeny from these crosses will be screened using PCR-based genotyping and flow cytometry to determine if elevated temperature leads to elevated haploid induction rate. These experiments could help establish experimental conditions that are optimal for using haploid induction lines for carrot breeding.
Impacts
What was accomplished under these goals?
During this project period, we focused our work on experiments related to the first two specific aims outlined in our grant proposal. The specific accomplishments that we have achieved are described after each specific aim listed below. Specific Aim 1. Evaluate the haploid-induction potential of carrot CENH3 mutant lines developed in our preliminary work In our previous experiments, we demonstrated how the CENH3 mutant carrot plants that we produced could cause chromosome elimination in progeny of crosses between those CENH3 mutants and wild-type carrots. In that prior work, we observed two progeny plants with chromosomal changes: one progeny was aneuploid and one was a tetraploid in which all of the chromosomes were inherited from the pollen donor in the cross, suggesting that genome elimination of the maternal genome had occurred. Although no haploid progeny were observed in those initial experiments, the results were consistent with the mutant CENH3 line causing chromosome elimination. During this reporting period, we analyzed the progeny produced by a new round of genetic crosses between these same CENH3 mutant lines and wild-type carrots. With this new round of screening, we tested 32 progeny plants and found that 5 of them had lost one copy of chromosome 7. We have performed follow-up testing on two of these progeny using flow cytometry and determined that one of these is a haploid plant and one is aneuploid. We will be testing the remaining three candidates soon using flow cytometry. These initial results have provided us with the first example of CENH3-based haploid induction in carrots. The rate with which we are observing aneuploid/haploid progeny is much higher in this second round of genetic crosses compared with the first experiments that we did. We have noted that the genetic crosses for these experiments were performed at substantially higher growth temperatures that the first round that led to less frequent aneuploidy. The explanation for this situation is that the first round of crosses were performed in the greenhouse in winter and the second round was done in the summer. The air temperature in the greenhouses that we use in Wisconsin for these experiments is significantly impacted by the outdoor temperature, so we know that plants growing in the winter experience a significantly cooler growth environment than those grown in the summer in these greenhouses. Because it has been shown in Arabidopsis that elevated growth temperature can significantly increase the rate of haploid induction with CENH3 mutant lines, we hypothesize that the higher rate of aneuploid/haploid progeny that we have observed for this second round of crosses is due to the elevated temperature. We plan to directly test this hypothesis in the next project period. Specific Aim 2. Generate additional CENH3 mutations to optimize haploid-induction rate. The postdoctoral scientist hired to work on this project has produced three new guide RNAs that allow us to introduce amino acid substitutions into additional sites in the coding sequence of the carrot CENH3 gene. We have regenerated gene edited carrot plants from protoplasts that had been transformed with the cytosine base editor construct developed in our previous work expressing these new sgRNAs and have identified several homozygous, gene edited lines. We are currently growing these gene edited plants and will move forward with testing their haploid induction potential to determine if any of the new mutations lead to improved haploid induction compared with our current CENH3 mutatns. For these experiments we will carefully control the growth temperature of the plants during the genetic crossing period to allow comparison between the haploid induction rate of these new mutants compared with our existing lines.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Yarra R, Krysan PJ. An SpG-Cas9-based cytosine base editor expands the scope of genome editing in carrot plants. Plant Cell Rep. 2024 Mar 5;43(3):82. doi: 10.1007/s00299-024-03173-3. PMID: 38441656.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Meyer CM, Goldman IL, Krysan PJ. Chromosome-level changes and genome elimination by manipulation of CENH3 in carrot (Daucus carota). Front Plant Sci. 2023 Nov 15;14:1294551. doi: 10.3389/fpls.2023.1294551. PMID: 38034555; PMCID: PMC10684906.
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The main target audience for this work is undergraduate students, graduate students, postdoctoral scientists, industry scientists, and faculty members. During this first year of the project, we presented a research seminar at the University of Wisconsin-Madison campus that provided an update on our work towards creating haploid inducers for carrot breeding. This seminar was attended by undergraduate students, graduate students, postdoctoral scientists and faculty members interested in plant biology, plant breeding, and plant cell and molecular biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Theexperimental work for this project is being carried out by a post-doctoral scientist who was hired for this project. The project provides them with the opportunity to gain experience carrying out gene editing in carrot and testing haploid induction. The project also provides the opportunity to present their work through seminar presentations. How have the results been disseminated to communities of interest?We have submitted a manuscript describing our initial observation of haploid induction caused by mutation of carrot CENH3. This manuscript is currently under review at Frontiers in Plant Science. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue to produce additional gene edited carrot plants carrying novel mutations in the CENH3 gene in order to test the haploid induction efficiency of these novel alleles. As novel mutant lines are produced, they will be put into a genetic crossing pipeline in which each mutant line will be crossed with a wild-type crossing partner. Progeny from each CENH3 mutant line will then be screened by PCR-based genotyping to identify candidate plants for further testing to determine if they are haploids. Using this approach, we will document the haploid induction potential of a number of new CENH3 mutant lines.
Impacts
What was accomplished under these goals?
During this project period we have focused our work on experiments related to the first two specific aims outlined in our grant proposal. The specific accomplishments that we have achieved are described after each specific aim listed below. Specific Aim 1. Evaluate the haploid-induction potential of carrot CENH3 mutant lines developed in our preliminary work. In our previous work, we produced 19 carrot lines carrying mutations in the CENH3 gene that encode amino acid substitutions in the CENH3 protein. We performed genetic crosses between these mutant lines and wild-type carrot plants in order to test the ability of these CENH3 mutants to cause haploid induction. Seed was collected from the CENH3 mutant lines, and the progeny were tested by PCR-based genotyping to search for evidence of haploid-induction. Using this approach, we identified two progeny plants that had alterations in their chromosomes. Follow-up testing via flow cytometry and whole-genome sequencing revealed that one of these progeny was an anueploid plant that only had a single copy of chromosome 7. It has been widely reported that haploid inducer lines in other plant species frequently cause this type of aneuploid progeny to be produced, so this result is consistent with the CENH3 mutant carrot plant affecting chromosome functions. Analysis of the second progeny plant indicated that all of its chromosomes were inherited only from the wt parent used in the cross, indicative of haploid induction. Surprisingly, flow cytometry indicated that this plant was a tetraploid. Our current hypothesis is that this plant initiated as a haploid, but early in development two rounds of chromosome doubling occurred which led to the tetraploid state that we observed in the mature plant. We documented these results in a manuscript that is currently under review in the journal Frontiers in Plant Science. This initial study provides the first evidence that mutation of carrot CENH3 can lead to haploid induction. Specific Aim 2. Generate additional CENH3 mutations to optimize haploid-induction rate. The postdoctoral scientist hired to work on this project has produced three additional guide RNAs that allow us to introduce amino acid substitutions into additional sites in the coding sequence of the carrot CENH3 gene. We have validated the efficiency of these sgRNAs in the editing reaction and are currently regenerating carrot plants from protoplasts that had been transformed with the cytosine base editor construct developed in our previous work expressing these new sgRNAs. We also produced an sgRNA sequence that will target a stop codon into the CENH3 gene in order to allow us to test the haploid induction potential of a plant heterozygous for a null allele of CENH3. We are currently regenerating carrot plants from those transformations as well. Once we have identified regenerated carrot plants carrying novel mutations, we will move forward with testing their haploid induction potential.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Meyer CM, Goldman IL, Krysan PJ. Chromosome-level changes and genome elimination by manipulation of CENH3 in carrot (Daucus carota). Frontiers in Plant Science. Under Review.
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