Progress 12/01/23 to 11/30/24
Outputs
Target Audience:The audiences targeted by this project include researchers working in basic and applied aspects of wheat research. Our results will be also of interest for researchers working on the effects of plant development on plant improvement as well as for researchers working in the regulation of flowering and plant age. This project also targets wheat breeders interested in grain yield improvement and growers interested in increases in wheat grain yield potential.This project is of interest also for breeders interested in forage grass species since the reduction of the juvenile phase is expected to increase biomass production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunities to five promising young scientists. Qiujie Liu (female), was the first postdoc in this project but she left in February 2023 because she got a tenure track professor position. She continues to be involved in the project from her own laboratory.Dr. Dubcovsky hired Lili Zhang (female) to replace Qiujie and she joined the lab in November 2022, which gave her the opportunity to overlap with Qiujie for four months. This facilitated the training of Lili Zhang on the materials developed in the project and the different laboratorytechniques required to complete the proposed objectives. The transition between the two postdocs was also facilitated by the overlap of Cynthia Zhou with both Qiujie and Lili. Cynthia is a UC Davis student that graduated in 2022 and that workedfull time in this project as Junior Specialist until her departure in May 2023.Cynthia was well trained by Qiujie and Lili, and her lab experience and strong recommendation letters, helped her to get in her desired graduate school program at the Johns Hopkins University. Lili Zhang hired a new undergraduate student, Xinyue Zhang (female), who is now working in this project supervised by Lili Zhang and Jorge Dubcovsky. Dr. Dubcovsky provided both Qiujie and Lili opportunitiesto mentor graduate and undergraduate students to help them develop their leadership and people management skills.Both Qiujie and Lili have been excellent mentors of the students under their responsibility. Finally, Dr. Joshua M Hegarty (male), a project scientist at UCD, is responsible for the field experiments in this project. He completed the first field evaluation of the lines in 2024. He was trained as a graduate student and as a postdoctoral researcher in small grains breeding by Dr. Dubcovsky. The overlaps between Qiujie, Lili and Cynthia facilitated a smooth personnel transition. Thishelped Lili to make significant progress in her first year in the project, as described in detail in the accomplishment section in this report. Dr. Dubcovsky is working now with Qiujie and Lili in a manuscript from this project, to contribute to their training in writing scientific articles, logical thinking, and statistical analyses. Dr. Dubcovsky taught Experimental Design for 20 years and provides students and postdocs in his lab with good training in this area. How have the results been disseminated to communities of interest?Results generated from this grant were published in a paper: "LEAFY and WAPO1 jointly regulate spikelet number per spike and floret development in wheat .", in the prestigious journal Development in August 2024. We also submitted the first spatial transcriptomics and single-cell RNA-seq study of wheat spike development, including all the wheat SPL genes characterized in this project. "Spatial and single-cell expression analyses reveal complex expression domains in early wheat spike development", which is under review in "Genome Biology" A preprint is available in bioRxiv https://doi.org/10.1101/2025.02.15.638402 This project was also discussed at the WheatCAP project meeting in January 2024 and 2025, where most of the US public wheat breeders were present. We also presented this project to the California Wheat Commission and to the wheat growers at the UC Davis Field Day as an example of our strategies to improve early growth, biomass and grain yield. The California wheat grower have requested the UCD breeding program to develop dual purpose wheat varieties that can be used both for forage and for grain for the bread market, to give them more flexibility to commercialize their wheat. Therefore, they are very excited about the preliminary results presented in this report, which suggest that the mutations generated in this project can be used to improve early growth and wheat biomass production. What do you plan to do during the next reporting period to accomplish the goals?Expand the miR156 knock-out combinations:During the fourth year of the project, we will complete the development of a line with combined loss-of-function mutations in all wheat miR156 loci. In particular, we will continue to focus on themiR156b,clocus, which includes three tandemly duplicated copies designatedmiR156b,c-p1,miR156b,c-p2andmiR156b,c-p3. We have developed two new guide RNAs targeting all the three tandem copies, which have been already transformed into the miR156-4 mutant. Most recently, we identified a new T0 mutant with deletion on miR156b,c-p1A, and miR156b,c p1-p2-p3Bon miR156-4 background which carries mutations in the two homeologs of themiR156d,miR156e,miR156g, andmiR156igenes. This means that we will have a line including mutations in 11 miR156 loci. We will cross itwith Kronos,mir156-2, andmir156-4 mutant respectively to remove the cas9 and generate different mutant combinations. Additionally, we will continue to identify new mutants with deletion in the threemiR156b,ccopies in both genomes to get a line including mutations in 13 miR156 loci. Once this line is obtained, we will also cross itwith Kronos,mir156-2, andmir156-4 mutant respectively to remove the cas9 and generate different mutant combinations. Expand the molecular characterization of miR156 knock-out combinations:We willexpand the molecular characterization of the combined mutantsmiR156-1,miR156-2,miR156-3,miR156-4to include miR156-5 which carries 9 mutated miR156 loci. We will quantify miR156 expression in roots tips and leaves (1stand 3rdleaf) in both hydroponic tanks and greenhouse experiments (for the aerial part only). We will evaluate mature miR156 expression levels by stem-loop qRT-PCR using protocols published before by our laboratory. In the same cDNAs, we will quantify expression levels ofSPL3, SPL4, SPL13andSPL23to determine if specific miR156 loci target specificSPLgenes. Expand the combinations of differentrSPLs:We will continue to screen the F4populations developed from the crosses between rSPL-A3with a plant carryingrSPL-A13 rSPL-A4andrSPL-A23to generate and complete all double, triple, and quadruple combinations of differentrSPLmutations. In addition, wewill explore the effect of the four SPL genes using differentrSPLmutant combinations on the expression of critical wheat flowering and tiller development genes. Phenotypic characterization: In the next phenotypic experiments we will includethenew miR156,rSPLandsplmutant combination developed during the previous year. In addition to the traits measured in 2024 (described in the accomplishment section of this report), we will include biomass measurements of roots and aerial parts, and will determine grain number per spike, grain weight, grain size, and grain yield per spike. Hydroponic experiments: In the next hydroponic experiments we will includethenew miR156,and different rSPL combination developed during the previous year. The main focus of thehydroponic experiments will be the evaluation of the effects of these mutations on primary root length (non-destructive) and total root biomass (destructive) at four different time points (7, 14, 21 and 28 DAG). For the shoots, we will measuretiller number, leaf number, leaf length and width in the non-destructive experiments and biomass in the destructive ones. Greenhouse experiments: The aerial phenotypes will be analyzed in more detail in the greenhouse experiments, which will include ten2.2-gallon pots per genotype each including 8 plants. Plants will be randomized every two weeks. As inthe hydroponic experiments, we will perform non-destructive and destructive experiments to evaluate biomass. In the non-destructive experiments, we will measure leaf blade length and width of the first, third, fifth and flag leaves, diameter and length of the second internode, total tiller number, fertile tiller number, heading date, spikelet number in the main spike, average grain weight and size (using a MARVIN seed analyzer), and grain yield per spike. The non-destructive experiments will also serve as a seed increase for field experiments. For mutants showing differences in tiller number, we will perform a separate experiment to test plant performance under different planting densities. The destructive experiments will be also performed in 2.2-gallon pots with 8 plants per pot and 10 pots per genotype. All the aerial biomass will be harvested and total wet and dry weight will be determined. Separate destructive experiments will be harvested at 7 days, 21days, flag leaf and milky ripe seed stage (ifenough seed is available).
Impacts
What was accomplished under these goals?
Objective 1: Manipulate miR156 dosage by combining deletions in miR156 loci. We generated 5 different homozygous combinations of edited miR156 loci in the tetraploid wheat variety Kronos, designated as: miR156-1: 2 edited loci (miR156b,c-p3). miR156-2: 2 edited loci (miR156g). miR156-3: 6 edited loci (miR156b,c-p3, miR156g, miR156i). miR156-4: 7 edited loci (miR156d, miR156g, miR156e, miR156i). miR156-5:9 edited loci (miR156b,c-p3, miR156d, miR156e, miR156g, miR156i). The miR156b,c locus in wheat includes three precursors in tandem, designated as miR156b,c-p1, -p2 and -p3. Using CRISPR in a miR156-4 background, we obtained T0 plants with a complete loss of miR156b,c-p1 in the A-genome and of miR156b,c-p1, -p2 and -p3 in the B-genome. Objective 2. Generate mutations in the miR156 binding sites of SPL3, SPL4, SPL13 and SPL23. Mutations in the miR156 binding site of the SPL generate dominant-mutants designated resistant SPLs of rSPLs. We obtained rSPLs mutants for all four genes and generated populations segregating for the four genes. In these populations, we identified plants homozygous for the different mutant and wildtype allele combinations. Furthermore, we obtained plants carrying rSPL-A3 and rSPL-B3 and crossed them with the rSPL-A4 rSPL-A13 rSPL-A23 triple mutant to generate double, triple, and quadruple mutants. More recently, we screened F2 and F3 populations of a quadruple mutant and obtained homozygous plants for different allele combinations. In addition to the dominant rSPL mutants, we generated SPL loss-of-function mutants using CRISPR and multiple guide RNAs. This year we crossed plants segregating for mutations in spl-A14, spl-A16, spl-B16, spl-A17, spl-B17, and spl-B23 with the spl-A13 spl-B13 double mutant. We are currently selecting different combinations. Objective 3. Quantify the effects of the different miR156 and SPLs mutant combinations on root and shoot growth. 3.1. Effects of the different miR156 mutant combinations We evaluated the phenotypic effect of different miR156 mutant combinations relative to the wildtype sister lines in field experiment and greenhouse experiments. Field experiments: In 2024, we analyzed our first field experiment with multiple miR156 including miR156-1, -2, -3 and their responding wild type. For these rows, we determined the number of fertile tillers, plant height, heading time, SNS, grain number per spike, average grain weight, grain size and grain weight per spike. We also evaluated canopy spectral data including vegetative indexes NDVI and NDRE, and water status index NWI3 (proxy for root depth). During the early season, miR156-1 and miR156-3 exhibited significantly higher NWI3 levels compared to the wild type (13.6-28.8%), suggesting that miR156-1 and miR156-3 might contribute to better-developed root systems. Besides, miR156-1 exhibited a longer spike and higher SNS (4.2-7.9%). miR156-3 was associated with a thicker stem (10.4%), and longer spikes (4.7%). Meanwhile, miR156-2 showed significantly lower tiller number compared to the wild type (8.2%), there was no differences in miR156-1 and miR156-3. Additionally, miR156-2 showed significantly fewer spikelets per spike (8.6%) and reduced grain weight (10%). Greenhouse pot experiment: The previous small and large pot experiment showed increases in leaf width, length and estimated leaf area in all four mutants except in miR156-1.The mutants also headed earlier and had less tillers. This year we characterized the phenotypes of miR156-4, MIM156 and wild type control. The MIM156 mutant exhibited significant reductions in total leaf number (by 20.0%), tiller number (by 21.7%), spike length (by 6.6%), and spikelet number per spike (SNS, by 8.6%), while also showing an earlier heading time by 6.2 days. Similarly, the miR156-4 variant demonstrated significant decreases in total leaf number (by 13.9%) and advanced heading time by 2.3 days. These findings highlight the large phenotypic changes associated with miR156-4 and MIM156 compared to the wild-type. In summary, the miR156 mutants exhibited earlier heading time and larger leaves. The different effects in the different miR156 mutant combinations suggest that different loci may regulate plant development in different way. 3.2. Effects of the different SPLs mutant combinations Resistant SPL mutants: To study the effects of the SPLs on heading time, we focused on the SPLs expressed in the vegetative tissues where the FT1 florigen is produced. We generated lines with mutations in miR156 binding sites, designated as rSPL-A3, rSPL-B3, rSPL-A4, rSPL-A13, rSPL-B13, and rSPL-A23. We evaluated the phenotypic effect of different rSPLs mutant combinations relative to the wild-type sister lines in field experiment, growth chamber experiments, greenhouse experiments, and hydroponic experiments. Field experiments: In 2024 we completed our first field experiment including multiple rSPLs dominant mutants and their corresponding wild type.During the early season, rSPL-A23 and rSPL-B13 exhibited significantly higher NWI3 indexes compared to the wild type (4.4% and 11.36%, respectively), suggesting that these mutants possess a better-developed root system. Additionally, rSPL-A23 headed 0.7 days earlier and had more spikelets per spike (4.0%), more seeds per spike (19.9%), and greater seed weight per spike (16.4%). In contrast, rSPL-A3 displayed a shorter spike length (4.9%), fewer spikelets per spike (2.8%), and fewer seeds per spike (9.5%). Meanwhile, rSPL-B13 exhibited larger seeds, with an increase of 2.8%. Growth chamber experiment: We evaluated the effect of rSPL-A3 on heading time in BC3F2 plants. Homozygous mutants of rSPL-A3 headed 1.9 d earlier than the wild type and had 3.5% fewer leaves. We repeated the experiment in the next generation. Homozygous mutants of rSPL-B3 headed 1.8 d earlier than the wild type but showed no significant difference in leaf number. Plants carrying simple dominant alleles for rSPL-A4 and rSPL-A13 headed 3-4d earlier than the wild type, whereas those carrying both resistant alleles headed 5.8 d earlier, indicating redundancy between the two alleles. No significant differences were detected for leaf number, tiller number and plant height. Finally, we generated CRISPR mutants for both rSPL-A13 and rSPL-B13. Both homozygous mutants headed earlier than their respective wildtype sister lines (3.0 and 2.4 d, respectively). In summary, these results confirmed the significant effect of the rSPL mutants on the acceleration of wheat heading time. Greenhouse experiment: we screened an F3 population segregating for rSPL-A4, rSPL-B3, rSPL-A13 and rSPL-A23 quadruple mutant, and we obtained homozygous plants for the different mutant and wildtype allele combinations. The different rSPL combinations exhibited significantly earlier flowering (1.9 to 6.5d) and reduced leaf number (5.9% to 18.2%) compared to the wild type. The triple mutant, rSPL-A4, rSPL-B3, and rSPL-A13, exhibited the earliest flowering (6.5d) and the largest reduction in leaf number (1.8). No significant differences were detected among these mutants for spike length, effective tiller number, or flag leaf length. Double, triple and quadruple dominant mutant for these three genes exhibited 7.9-10.2% reductions in SNS. Moreover, different mutant combinations exhibited longer (19.4% to 26.2%) and narrower (1.5% to 14.7%) leaves. These findings are consistent with the observations in MIM156 mutants, including earlier flowering time, fewer leaves, reduced SNS, and longer juvenile leaves. In summary, our results indicate that: The earlier heading time and less SNS of the wheat miR156 mutants is mediated by the upregulation of multiple SPL genes. Wheat SPL3, SPL4, SPL13, and SPL17 promote heading time. The functional SPL14 and SPL17 increase SNS in wheat. miR156 mutants and rSPL4 dominant mutants increase wheat primary root length.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Paraiso F, Zhang J, Lin H, Li C, Woods DP, Lan T, Debernardi JM, Joe A, Dubcovsky J (2024) LEAFY and WAPO1 jointly regulate spikelet number per spike and floret development in wheat. Development 151:dev202803.
- Type:
Peer Reviewed Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Xu X, Lin H, Zhang J, Burguener G, Paraiso F, Tumelty C, Li C, Liu Y, Dubcovsky J (2025) Spatial and single-cell expression analyses reveal complex expression domains in early wheat spike development. bioRxiv https://doi.org/10.1101/2025.02.15.638402 Submitted to Genome Biology
|
Progress 12/01/22 to 11/30/23
Outputs
Target Audience:The audiences targeted by this project include researchers working in basic and applied aspects of wheat research. Our results will be also of interest for researchers working on plant development effects on plant improvement as well as for researchers working in the regulation of flowering and of the plant juvenile phase. This project also targets wheat breeders interested in grain yield improvement and growers interested in increases in wheat grain yield potential.This project is of interact also for breeders interested in forage grass species since the reduction of the juvenile phase is expected to increase biomass production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunities to five promising young scientists. Qiujie Liu (female), was the first postdoc in this project but she left in February 2023 because she got a tenure track professor position in China. She continues to be involved in the project from her own laboratory. Dr. Dubcovsky hired Lili Zhang (female) to replace Qiujie and she joined the lab in November 2022, which gave her the opportunity to overlap with Qiujie for four months. This facilitated the training of Lili Zhang on the materials developed in the project and the different laboratory techniques required to complete the proposed objectives. The transition between the two postdocs was also facilitated by the overlap of Cynthia Zhou with both Qiujie and Lili. Cynthia is a UC Davis student that graduated in 2022 and that workedfull time in this project as Junior Specialist until her departure in May 2023. Cynthia was well trained by Qiujie and Lili, and her lab experience and strong recommendation letters, helped her to get in her desired graduate school program at the Johns Hopkins University. Lili Zhang hired a new undergraduate student, Haoyue Dong (female), who is now working in this project supervised by Lili Zhang and Jorge Dubcovsky. Dr. Dubcovsky provided both Qiujie and Lili opportunitiesto mentor graduate and undergraduate students to help them develop their leadership and people management skills.Both Qiujie and Lili have been excellent mentors of the students under their responsibility. Finally, Dr. Joshua M Hegarty (male), a project scientist at UCD, is responsible for the field experiments in this project. He was trained by as a graduate student and as a postdoctoral researcher in small grains breeding by Dr. Dubcovsky. The overlaps between Qiujie, Lili and Cynthia facilitated a smooth personnel transition. This helped Lili to make significant progress in her first year in the project, as described in detail in the accomplishment section in this report. Dr. Dubcovsky is working now with Qiujie and Lili in a manuscript from this project, to contribute to their training in writing scientific articles, logical thinking, and statistical analyses. Dr. Dubcovsky taught Experimental Design for 20 years and provides students and postdocs in his lab with good training in this area. How have the results been disseminated to communities of interest?Results generated from this grant were published in a bioRxiv paper in 2023: "LEAFY interacts with WHEAT ORTHOLOG OF APO1 to regulate spikelet number per spike in wheat.". This paper has been submitted to Science Advances and is currently under review. This project was also discussed at the WheatCAP project meetings, where most of the US public wheat breeders were present. We also presented this project to the California Wheat Commission and to the wheat growers at the UC Davis Field Day as an example of our strategies to improve early growth, biomass and grain yield. The California wheat grower have requested the UCD breeding program to develop dual purpose wheat varieties that can be used both for forage and for grain for the bread market, to give them more flexibility to commercialize their wheat. Therefore, they are very excited with the preliminary results presented in this report, which suggest that the mutations generated in this project can be used to improve early growth and wheat biomass production. What do you plan to do during the next reporting period to accomplish the goals?Expand the miR156 knock-out combinations:During the third year of the project, we will complete the development of a line with combined loss-of-function mutations in all wheat miR156 loci. In particular, we will continue to focus on the miR156b,c locus, which includes three tandemly duplicated copies designated miR156b,c-p1, miR156b,c-p2 and miR156b,c-p3. We have developed two new guide RNAs targeting all the three tandem copies, which have been already transformed into the miR156-4 mutant. miR156-4 carries mutations in the two homeologs of themiR156d, miR156e, miR156g, and miR156igenes, so if we are successful in deleting the three miR156b,c copies in both genomes, we will have a line including mutations in 13 miR156 loci. Once this line is obtained, we will cross itwith Kronos, mir156-2, and mir156-4 mutant respectively to remove the cas9 and generate different mutant combinations. Expand the molecular characterization of miR156 knock-out combinations:We willexpand the molecular characterization of the combined mutantsmiR156-1,miR156-2,miR156-3,miR156-4to include miR156-5 which carries 9 mutated miR156 loci. We will quantify miR156 expression in roots tips and leaves (1st and 3rd leaf) in both hydroponic tanks and greenhouse experiments (for the aerial part only). We will evaluate mature miR156 expression levels by stem-loop qRT-PCR using protocols published before by our laboratory. In the same cDNAs, we will quantify expression levels ofSPL3, SPL4, SPL13andSPL23 to determine if specific miR156 loci target specific SPL genes. Expand the combinations of differentrSPLs:We will screen the F2populations segregating forrSPL-A3 andrSPL-B3 to identify homozygous double mutants and their respective wildtype sister lines. We will also screen the two F2population developed from the crosses between the A- and B-genome homeologs of rSPL3 with a plant carrying rSPL-A13 rSPL-A4 and rSPL-A23 to generate lines with double, triple, and quadruple combinations of differentrSPL mutations. In addition, wewill explore the effect of differentSPLmutant combinations on the expression of critical wheat flowering and tiller development genes. Phenotypic characterization: In the next phenotypic experiments we will includethenew miR156,rSPLand spl mutant combination developed during the second year (e.g. the miR156-5 mutant with 9 mutations). In addition to the traits measured in 2023 (described in the accomplishment section of this report), we will include biomass measurements of roots and aerial parts, and will determine grain number per spike, grain weight, grain size, and grain yield per spike. Hydroponic experiments: The main focus of thehydroponic experiments will be the evaluation of the effects of these mutations on primary root length (non-destructive) and total root biomass (destructive) at four different time points (7, 14, 21 and 28 DAG). For the shoots, we will measuretiller number, leaf number, leaf length and width in the non-destructive experiments and biomass in the destructive ones. Greenhouse experiments: The aerial phenotypes will be analyzed in more detail in the greenhouse experiments, which will include ten2.2-gallon pots per genotype each including 8 plants. Plants will be randomized every two weeks. As inthe hydroponic experiments, we will perform non-destructive and destructive experiments to evaluate biomass. In the non-destructive experiments , we will measure leaf blade length and width of the first, third, fifth and flag leaves, diameter and length of the second internode, total tiller number, fertile tiller number, heading date, spikelet number in the main spike, average grain weight and size (using a MARVIN seed analyzer), and grain yield per spike. The non-destructive experiments will also serve as a seed increase for field experiments. For mutants showing differences in tiller number, we will perform a separate experiment to test plant performance under different planting densities. The destructive experiments will be also performed in 2.2-gallon pots with 8 plants per pot and 10 pots per genotype. All the aerial biomass will be harvested and total wet and dry weight will be determined. Separate destructive experiments will be harvested at 7, 21, flag leaf and milky ripe seed stage (ifenough seed is available). Field experiments: In November 2023, we planted our first filed experiment including multiple miR156 and rSPL mutants using 1 m rows as replications, and a variable number of replications determined by the available seeds. For these rows, we will determine number of plants and randomly select 10 individual plants (subsamples) for which we will determine the number of fertile tillers, number of leaves in the main tiller, plant height, heading time, SNS, grain number per spike, average grain weight, grain size using the MARVIN seed analyzer, and grain weight per spike. Values for each row will be averaged and rows will be used as replicates in the statistical analyses. Seeds from these rows will be used to plant a small plot experiment in November 2024 to evaluate biomass and grain yield. All field experiments will be conducted at the University of California field station in Davis, CA (38°32'N, 121°46'W), which has deep Yolo loam soils (fine-silty, mixed, superactive, non-acid, thermic Mollic Xerofluvent). The experiments will be planted in November and harvested in June. Fertilization will include two application of 112 kg ha-1 N at pre-planting and tillering. A broadleaf herbicide will be applied in mid-February and flood irrigation will be applied as needed.
Impacts
What was accomplished under these goals?
Objective 1: Manipulate miR156 dosage by combining deletions in miR156 loci. We generated 5 different homozygous combinations of edited miR156 loci in the tetraploid wheat variety Kronos, designated as: miR156-1: 2 edited loci (miR156b,c-p3)1. miR156-2: 2 edited loci (miR156g). miR156-3: 6 edited loci (miR156b,c, miR156g, miR156i). miR156-4: 7 edited loci (miR156d, miR156g, miR156e, miR156i). miR156-5:9 edited loci (miR156b,c, miR156d, miR156e, miR156g, miR156i). 1 Mutations in A and B genomes except miR156e whichonly has mutations in the A genome. The miR156b,c loci in wheat include three precursors in tandem, designated as miR156b,c-p1, -p2 and -p3. Their tight linkage precludes the combination of independent mutations by recombination. Therefore, we generated transgenic plants in the miR156-4 mutant background using pairs of guide-RNAs designed to generate deletions including the duplicated copies. Objective 2. Generate mutations in the miR156 binding sites of SPL 3, SPL4, SPL13 and SPL23. We obtained resistantSPL (rSPLs) mutants for all four genes, and generated populations segregating for rSPL-A4 and rSPL-A13, forrSPL-A13 and rSPL-A23, and for all three genes. In these populations, we identified plants homozygous for different mutant and wildtype allele combinations. More recently, we obtained plants with rSPL-A3 and rSPL-B3 and crossed them with the rSPL-A4 rSPL-A13 rSPL-A23 triple mutant to generate double, triple, and quadruple mutants. In addition to the rSPL mutants, we generated SPL loss-of-function mutants using CRISPR and multiple guide RNAs. We identified 124 plants segregating for mutations in SPL-A14, SPL-A16, SPL-B16, SPL-A17, SPL-B17, and SPL-B23, and weused them to estimate their effects on different traits and their epistatic interactions. Objective 3. Quantify the effects of the different miR156 and SPLs mutant combinations on root and shoot growth. 3.1. Effects of the different miR156 mutant combinations We evaluated the phenotypic effect of different miR156 mutant combinations relative to the wildtype sister lines in hydroponic conditions, and in two greenhouse experiments. Hydroponic experiment: In this experiment we evaluated the effect of the miR156 mutations on primary root length at 5, 7, 14, 21 and 28 days after germination (DAGs). We observed highly significant root length increases in miR156-2, -3, and -4 relative to the wildtype sister lines. These increases varied from 20 to 40% in miR156-4 (14 to 28 DAG), 17 to 20% in miR156-3 (5 to 28 DAG), and 28% to 11% in miR156-2 (5 to 14 DAG). The different significant timepoints in the different miR156 mutant combinations suggest that different loci may regulate different stages of root development. For the aerial part, all 3 mutants showed significantly wider and longer leaves than the wildtype, that were reflected in significant increases in estimated leaf area. In miR156-4 these increases varied from 28% for the 1st leaf to 52% for the 4th leaf. The miR156-3 plants showed smaller relative differences, which were significant only for the 4th (17%) and 5th leaves (35%). The increases in leaf area in the miR156-2 mutants (average 16%) were similar to those in miR156-3 but were significant from the 1st to the 4th leaf. In summary, the miR156 mutants showed significantly longer roots and larger leaves than the wild type, with larger changes in the plants with more mutated miR156 loci. Greenhouse small pot experiment: This experiment showed increases in leaf width, length and estimated leaf area for at least one of the leaves in all four mutants except in miR156-1. Combined mutants miR156-2, -3, and -4 also showed significant reductions in the total leaf-number (1.2 to 3.6 less leaves), earlier heading time (1.8 to 6.7 days), and increased SNS (1.2 to 2.8 more spikelets per spike). Greenhouse large pot experiment: For miR156-3, miR156-4 and the wildtype sister-line, we performed a second greenhouse experiment in large 2.2-gallon pots with 8 plants per pot (subsamples) and 8 to 12 pots per genotype. Measurements in the 5th leaf showed significant increases in estimated leaf area (24 to 56%) in the mutant lines. Compared with the wildtype, the miR156-3 and miR156-4 mutants showed 1.3 and 1.5 less leaves, 1.2 and 0.8 less tillers, headed 4.0 and 4.7 days earlier, and showed a 21% and 23% increase in SNS, respectively. In summary, the miR156 mutants exhibited earlier heading time, had larger leaves, and more spikelets per spike. We increased seeds for miR156-1, miR156-2 and miR156-3 and planted them with their wildtype sister lines as 1 m rows in the field in winter 2023 to increase seeds for future yield experiments. 3.2. Effects of the different SPLs mutant combinations Resistant rSPL mutants: To study the effects of the SPLs on heading time, we focused on the SPLs expressed in the vegetative tissues where the FT1 florigen is produced. We generated lines with mutations in the miR156 binding sites, designated as rSPL-A3, rSPL-B3, rSPL-A4, rSPL-A13, rSPL-B13, and rSPL-A23. We intercrossed rSPL-A4 and rSPL-A13 and studied their phenotypic effect in a factorial design. Plants carrying only the rSPL-A4 or rSPL-A13 alleles headed 3.8 and 3.3 d earlier than the wild type, respectively (P < 0.001). Plants carrying both resistant alleles headed on average 5.8 d earlier than the wildtype suggesting some redundancy in the effects of the two alleles. No significant differences were detected between the wildtype and the single or combined mutants (Dunnett tests) for leaf number, tiller number and plant height. However, the rSPL-A4 rSPL-A13 double mutant showed 1.6 spikelets less than the wildtype (P < 0.01). We also evaluated the effect of rSPL-A4 on root development in a hydroponic experiment. We observed a highly significant increase in root length (15 to 24%) at all six time points from 5 to 21 DAG, which is consistent with the longer roots observed in the miR156 combined mutants. The miR156 target site in SPL13 is located in the 3' UTR, which facilitated the generation of CRISPR mutants in the target site without disrupting the reading frame. Both rSPL-A13 and rSPL-B13 homozygous mutants in the target site showed increased transcript levels and headed earlier than their respective wildtype sister lines (3.0 and 2.4 d, respectively). The rSPL-A23 mutant showed no significant differences in heading time, leaf number or tiller number relative to the wildtype. Loss-of function SPL mutants: We edited Kronos plants with guide RNAs targeting multiple SPLs and obtained 124 BC1F3 plants segregating for loss-of-function mutations in SPL-A14, SPL-A16, SPL-B16, SPL-A17, SPL-B17 and SPL-B23. A factorial ANOVA including these plants showed a highly significant reduction in SNS associated with SPL-A17 and SPL-B17. A simplified ANOVA using only the previous two genes and the related SPL-A14, revealed highly significant effects on SNS for the three genes and significant interactions. The SPL-A14 homozygous mutant was associated with a reduction of 2.3 spikelets per spikes, whereas the SPL-A17 and SPL-B17 mutants had 5.6 and 3.8 spikelets per spike less than the wildtype. The triple mutant showed 8.4 less spikelets than the wildtype (46% reduction). We also observed a slight but significant delay in heading time associated with SPL-A17 (3.2 d) and SPL-B17 (3.7 d). Based on these results, we concluded that SPL17 and SPL14 play important roles in spike development and a more limited role on heading time. In summary, our results indicate that The earlier heading time of the wheat miR156 mutants is mediated by the upregulation of multiple SPL genes. Wheat SPL3, SPL4, SPL13, and SPL17 promote heading time. The functional SPL14 and SPL17 increase SNS in wheat. miR156 mutants and rSPL4 dominant mutants increase wheat primary root length.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Paraiso, F., Zhang, J., Lin, H., Li, C., Woods, D.P., Lan, T., Debernardi, J.M., Joe, A. and Dubcovsky, J. (2023) LEAFY interacts with WHEAT ORTHOLOG OF APO1 to regulate spikelet number per spike in wheat. bioRxiv, 2023.2011.2002.565263.
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Progress 12/01/21 to 11/30/22
Outputs
Target Audience:The audiences targeted by this project include researchers working in basic and applied aspects of wheat research. Our results will be also of interest for researchers working on plant development effects on plant improvement as well as for researchers working in the regulation of flowering and of the plant juvenile phase. This project also targets wheat breeders interested in grain yield improvements and growers interested in increases in wheat grain yield potential. Changes/Problems:Genetic stocks: We have previously identified radiation mutants in hexaploid wheat that eliminated all three tandem copies of miR156b,c-p1+p2+p3 in both the A and B genomes and initiated their transfer to Kronos by back-crossing. We performed an exon-capture experiment to delimit the deleted regions. The results showed that the radiation mutations were very large (40 Mb for the A genome and 60Mb for the B genome). Unfortunately, the large size of these deletions and the large number of deleted genes may have negative effects on the viability of agronomic performance of the double mutants, so we decided to stop the work with the radiation mutants and intensify the CRISPR-Cas9 approach to eliminate this three tandem miR156 without affecting the flanking genes. We have already created a new CRISPR-Cas9 vectors to target the different copies of miR156b,c. Personnel: Postdoc Qiujie Liu has to return to China in May 2023 for family reasons, so we hired a new postdoc, Lili Zhang, who is already working in the project. The five-months overlap between the two postdoctoral scientists will facilitate a smooth transition, a proper training of the new postdoc, and an orderly and efficient transfer of all the material developed during the first year of the project. What opportunities for training and professional development has the project provided?This project has provided training opportunities to three promising young scientists. Qiujie Liu (female), is the postdoctoral scholar leading this project. This is her first postdoctoral training after her PhD completion at Rutgers University. Cynthia Zhou, is a recently graduated UC Davis student that is working full time in the project as Junior Specialist. Finally, Li Meinhold (male) is an undergraduate student working in this project. Qiujie Liu works as a direct supervisor of Cynthia Zhou and Li Meinhold, providing her the opportunity to develop her leadership and people management skills. Cynthia and Li have been trained in wheat phenotypic analyses using hydroponic culture systems. Cynthia has been trained in the mid-seq sequencing pipeline for CRISPR genotyping. Cynthia has been trained in RNA isolation and qRT-PCR technology. Qiujie has been trained in Chip-qPCR technology. Qiujie, Cynthia and Li have been trained in the KASPar genotyping method. Qiujie Liu presented her results in laboratory meetings, and is currently writing a manuscript describing the results she obtained from the SPL mutants. These activities contribute to her training in oral and written communication. How have the results been disseminated to communities of interest?Results generated from this grant were published in a PLoS Genetics paper in 2022: "MiR172-APETALA2-like genes integrate vernalization and plant age to control flowering time in wheat". This paper, published only seven months ago, has been viewed 2,354 times and downloaded 1,293 times documenting the interest in this project by the international research community. This project was also discussed at the WheatCAP project meetings, where most of the US public wheat breeders were present. We also presented this project to the California Wheat Commission and to the wheat growers at the UC Davis Field Day as an example of our strategies to improve early growth and final grain yield. What do you plan to do during the next reporting period to accomplish the goals?Development of miR156 knock-out combinations: During the second year of the project, we will complete the development of loss-of-function mutations in all the miR156 loci. In particular, we will focus on the miR156b,c locus, which includes three precursors in tandem designated as miR156b,c-p1, miR156b,c-p2 and miR156b,c-p3. We have already detected mutations in miR156b,c-p3, so the focus will be on p1 and p2. For that, we have developed two new guide RNAs targeting all the three tandem copies, which have been already transformed into Kronos. As a backup, we will transform the miR156b,c-p3 mutant with guide RNAs targeting p1 and p2. Once the triple p1/p2/p3 mutant is obtained, we will combine them with mutants mir156-1, mir156-3 and mir156-4 by crossing. Molecular characterization of miR156 knock-out combinations: In the second year, we will evaluate the effect of the combined mutants mir156-1, mir156-2, mir156-3, mir156-4 developed in year one on miR156 dosage in roots tips and leaves (first and third) in both hydroponic tanks and green house. We will evaluate mature miR156 expression levels by stem-loop qRT-PCR using protocols published before by our laboratory. In the same cDNAs, we will quantify expression levels of SPL3, SPL4, SPL13 and SPL23. This will help us to determine if specific miR156 loci target specific SPL genes. Combined different rSPLs. In the second year, we will screen the F2 populations segregating for rSPL-A4+rSPL-B13 and for rSPL-A23+rSPL-B13 to identify homozygous double mutants and their respective wildtype sister lines. In addition, we will screen an F2 population from the cross rSPL-B13 // rSPL-A4 / rSPL-A23 to get lines with double and triple combinations of rSPL mutations. We will also cross the rSPL-B3 mutants with rSPL-A4, rSPL-B13 and rSPL-A23 to generate double mutants. We will explore the effect of different SPL mutant combinations on the expression of critical wheat flowering genes, as done for the single SPL mutants in year one. Phenotypic characterization: During the second year of the project we will increase seeds of the different combination of miR156 and rSPL mutants to initiate the phenotypic characterization in hydroponic and greenhouse experiments. The main focus of the hydroponic experiments will be the evaluation of the effects of these mutations on the root system. The aerial phenotypes will be analyzed in more detail in the greenhouse experiments, which will include 12 pots per genotype. Hydroponic experiments: Measurements will be taken at one, two, three and four weeks. For the roots, we will measure seminal root length for all the available plants (non-destructive). For the shoots, we will measure tiller number, leaf number, leaf blade length, leaf blade width and leaf sheath length for all plants and average the values per tank. Mutants and control averages will be compared using a Student t-Test and tanks as replications. Greenhouse experiments (GH): The main objective of these experiments is to evaluate the aerial parts and plant architecture. The mir156 and rSPL mutant combinations will be planted in one-gallon pots with 1 plant per pot. Twelve pots per genotype will be analyzed in a completely randomized design. Plants will be randomized every two weeks. We will measure leaf blade length and width of the first, third, fifth and flag leaves, diameter of the second internode, total tiller number, fertile tiller number, heading date, spikelet number in the main spike, grain weight and size (using a MARVIN seed analyzer), and grain yield per tiller. This experiment will also serve as a seed increase for the first field experiment. For mutants showing differences in tiller number, we will perform a separate experiment to test plant performance under different planting densities. In summary, the plans for year 2 follow closely what we described in the original proposal.
Impacts
What was accomplished under these goals?
Objective 1. Tools to manipulate miR156 levels We identified 5 loci in each of the wheat genomes that express miR156: miR156b,c (including precursors p1, p2 and p3 in tandem), miR156d, miR156e, miR156g, and miR156i. We used CRISPR editing to target all mature miR156 sequences using five guide RNAs. Using next-generation sequencing (NGS) of multiple transformation events in the tetraploid wheat Kronos, we identified CRISPR mutants that contain mutations in both the A and B homeologs for miR156b,c-p3, miR156g, miR156d, miR156e and miR156i. We designed genome specific primers for each of the different loci producing PCR products of <150 bp, which can be efficiently separated in 6%-PAGE gels. We then used these primers to combine the different mutant alleles. So far, we have obtained the following combined homozygous mutant lines without the CRISPR-Cas9 vector: mir156-1: 13-bp deletion in miR156b,c-A and 8-bp deletion in miR156b,c-B. mir156-2: 7-bp deletion in miR156g-A and 11-bp deletion in miR156g-B. mir156-3: 13-bp deletion in miR156b,c-A; 8-bp deletion in miR156b,c-B; 7-bp deletion in miR156g-A; 11-bp deletion in miR156g-B; 7-bp deletion in miR156i-A locus and a 3-bp deletion in miR156i-B. mir156-4: 7-bp deletion in miR156g-A; 5-bp deletion in miR156g-B; 13-bp deletion in miR156d-A; 5-bp deletion in miR156d -B; 7-bp deletion in miR156i-A, 3-bp deletion in miR156i-B locus and a 5-bp deletion in miR156e-A. In the previous CRISPR screen we obtained mutations only in the miR156b,c-p3 precursor, so we designed additional constructs (different guide RNAs) to knock out miR156b,c-p1 and miR156b,c-p2. We obtained 3 T0 plants that will be genotyped in year 2. Objective 2. Tools to manipulate SPL transcript levels. The activity of specific SPL genes was manipulated by introducing point mutations in their miR156 target site (henceforth resistant SPLs or rSPLs). Based on their expression profiles, we focused on SPL3, SPL4, SPL13 and SPL23. In our Kronos sequenced mutant population, we foundrSPL-A4 and rSPL-A23 mutations and backcrossed them into Kronos.These are dominant alleles, so mutations in one of the two genomes are likely sufficient to observe a phenotype. For SPL3, we did not find good mutations in Kronos, so we transferred mutations CAD1995 (rSPL-A3) and CAD1033 (rSPL-B3) from hexaploid wheat Cadenza into Kronos by backcrossing. We are currently increasing BC2F3 seeds for phenotypic analyses. For SPL13, we did not find good mutations, so we used CRISPR-Cas9 to target the miR156 target site, which is located in the 3' UTR of SPL13. Screening of 5 T0 plants for mutations identified 2 lines with large deletions in the miR156 binding site: rSPL-A13 (21-bp deletion) and rSPL-B13 (17-bp deletion). For rSPL-A13, we screened out the transgene in the F1s generated by crossing rSPL-A13 with Kronos, and for rSPL-B13 we screened the transgene out in the T1 progeny generated by self-pollination. So far, we have obtained the following rSPL mutant combinations: rSPL-A4+rSPL-A23 homozygous lines combining both mutations. rSPL-A4+rSPL-B13 double mutant. rSPL-A4+rSPL-A23 double mutant. rSPL-A4+rSPL-A23+ rSPL-B13 triple mutant. SPL3, SPL4, SPL13 and SPL23 expression was detected in Kronos third and fifth leaves by qRT-PCR. All four SPL genes showed increased expression with plant age (opposite to miR156). In addition, all four SPL genes showed significant down-regulation in the transgenic plant over-expressing Ubi::miR156 and up-regulation in the MIM156 transgenic plants (reduced miR156 levels), confirming their regulation by miR156. In root tips collected at 7 and 14 days after germination, we observed increased expression of SPL3 and SPL4 with plant age, but we also detected low expression and no differences with age for SPL13 and SPL23. We then compared the effect of the mutations in the miR156 target site on SPL expression in the three available tetraploid rSPL mutants and their respective sister lines(rSPL-A4, rSPL-B13, and rSPL-A23).). For rSPL-A4, we observed a 2-fold increase in SPL4 in the roots and a 4-fold increase in the third leaf relative to the wildtype control.For rSPL-B13, we observed a 2-fold upregulation in SPL13 in the third leaf relative to the control.However, we did not detect significant differences in SPL23 expression between rSPL-A23 mutants and wild type. This result could be explained by low expression of SPL23 in the third leaf or by a limited effect of the selected mutation in rSPL-A23 miR156 target site. Objective 3 Phenotypic characterization of the rSPL mutants. We increased the grains of rSPL-A4 (BC3F3), rSPL-B13 (T2), rSPL-A13 (F2), rSPL-A23 (BC1F3), and their respective wildtype sister lines to initiate a preliminary phenotypic characterization. These lines were evaluated in a growth chamber under long-day conditions. We measured total tiller number, heading date, and spikelet number in the main spike. For the single rSPL mutants, we did not observe significant differences in tiller number and spikelet number per spike, but, rSPL-A4, rSPL-A13 andrSPL-B13mutants headed two days earlier than their respective wildtype sister line. In year 2, we will evaluate the effects of the combined rSPL mutations. To explore the molecular mechanisms underlying rSPL-A4 and rSPL-A13 and rSPL-B13 earlier heading phenotype, we extracted RNA from the fifth leaf and checked the expression level of several known wheat flowering genes. We observed a significant upregulation of FT, FUL2 and miR172 in rSPL-A4 and rSPL-B13 mutants, indicating that SPL4 and SPL13 likely regulate wheat heading time through the regulation of these flowering genes. To evaluate the function of SPLs in a winter wheat background, we introduced rSPL-A4 and rSPL-B13 into a Kronos mutant (K2268), which carries a truncation mutation in VRN-A1 resulting in a strong vernalization requirement. We crossed rSPL-A4 (BC3F2) with K2268 and obtained an F2 segregating population, from which 200 plants are being evaluated for heading time. Approximately 50 plants are expected to have a winter growth habit, and among those we will evaluate the effect of rSPL4 on heading time. In addition, we crossed rSPL-B13 (T2) with K2268 to generate an F2 population to study its effect on heading time in a winter background. Phenotypic effects of miR156 Preliminary phenotypic characterization of mir156-4 combining mutations in seven miR156 loci demonstrated a significant effect of the mutations on reducing tiller number. Reduced tiller number and increased biomass production is one of the target phenotypes of this project, and a promising preliminary result. The effects of miR156 on wheat heading time were published in "PLoS Genetics" in 2022. In this paper, we showed that miR156 acts as an upstream regulator of miR172 integrating age information into the flowering pathway. In spring wheat, we showed that miR156 expression decreases with plant age, while miR172 expression increases, resulting in the downregulation of its targets, the APETALA2-like flowering repressors and in the acceleration of heading time. In winter wheat cultivars, however, the induction of miR172 and the downregulation of AP2L1 is decoupled from miR156, and the induction of VRN1 by vernalization is required in conjunction with the induction of miR172 for the repression of AP2L1 and the promotion of flowering. This study also showed that natural or induced mutations in the AP2L genes can be useful tools for fine-tuning wheat flowering time. This study characterized the interactions between the vernalization requirement and the conserved pathway that integrates plant age, expanding our understanding of the regulation of heading time in wheat.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Debernardi, J.M., D.P. Woods, K. Li, C. Li, J. Dubcovsky. 2022. MiR172-APETALA2-like genes integrate vernalization and plant age to control flowering time in wheat. PLoS Genetics, 18: e1010157. https://doi.org/10.1371/journal.pgen.1010157
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