Recipient Organization
DONALD DANFORTH PLANT SCIENCE CENTER
975 NORTH WARSON ROAD
ST. LOUIS,MO 63132
Performing Department
(N/A)
Non Technical Summary
Theaim of this project is to develop novel genetic methods to induce male sterility inwheatto facilitate production methods for hybrid seeds. The approach is based on preliminary data from studies on rice and maize that have identified pathways that, when perturbed, yield environmentally sterile phenotypes, i.e. are responsive to photoperiod or temperature. The project will target several pathways, with several targets thatgenerate abundant small RNAs. These two classes of RNAs are generated at the critical stages of cell fate setting and during meiosis in grass anther development. Disruption of their biogenesis or encoding loci in both rice and maize leads to conditional male sterility. The proposal exploits these observations to target and disrupt these RNAs in wheat and assess the consequences of this disruption on male sterility. The project will complement work on this pathway by targeting at least one other pathway known to generate a similar phenotype. The Pls' expertise covers small RNAs, plant genomics and targeted mutagenesis, bioinformatics, wheat genetics, cytogenetics and meiotic studies. The proposal is risky but there is preliminary data to suggest all aims are achievable. Success in this area could increase wheat yields by at least 10%, potentially more, representing an extra ~20 million metric tons of wheat in just the EU and US, grown with the same inputs and footprint.Currently there are few wheat hybrid cultivars grown, generated either based on chemical hybridisation agents or on cytoplasmic male sterility (CMS) systems. The major limitations of the widespread use of wheat hybrids are seed production and costs. The present proposal assesses an alternative route to generating hybrids. The broader impacts of the proposed project include the potential to substantially increase wheat yields with no added land area for production or chemical inputs. The work will advance insights into eukaryotic small RNAs, a field of significant breadth and activity with many fundamental discoveries from the plant kingdom. An important impact is on the training and advancement of post-docs who will be trained broadly in plant small RNA biology and genomics, reproductive biology, crop improvement, and computational methods. Academic collaborations with other labs are integral to the project and will extend the impact of the experimental, computational, and genomics techniques that are an integral part of the project.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Aim 1. Characterization of reproductive phasiRNAs & biogenesis components in wheatThe purpose of this aim is to characterize the complete set of reproductive phasiRNAs (21- and 24-nt) and their mRNA precursors in hexaploid wheat. We have substantial prior experience in maize and rice. Generating the baseline dataset on wheat reproductive phasiRNAs will require isolation of developmentally staged anthers (premeiotic, meiotic to maturing microspores), and sequencing of both small RNA and mRNA (for phasiRNA precursors). Staged materials have been offered (no strings) by Dr. Tim Kelliher (Syngenta) in the genotype "Fielder". All sequencing data will be handled using our long-established pipelines, visualization system & web browser, and analysis tools. This informatics system allows us to rapidly identify phased loci and quantify abundance differences across developmental stages. We anticipate annotating hundreds of lncRNA precursors, as well as miR2118 and miR2275 precursors - data required for aim 2 & 3. We will identify all small RNA biogenesis components encoded in the wheat genome, using mRNA data to refine gene models and expression dynamics for Dicers (DCLs), Argonautes (AGOs), etc.Aim 2. Alterations in reproductive phasiRNA biogenesis and carbon-starved anther pathway in wheat as a path to conditional male sterilityThis objective is the major component and highest priority of the project. We have several subaims to maximize the likelihood of success in ultimately achieving conditional male sterility in wheat. One subaim is to disrupt the biogenesis of (i) the 21-nt premeiotic phasiRNAs, via deletion of genomic loci that give rise to the triggers of the 21-mers, miR2118, or (ii) the 24-ntmeiotic phasiRNAs, a more straightforward approach given several biogenesis components unique to this pathway. Additional subaims are to (iii) perturb a small number of premeiotic phasiRNAs, or (iv) to target a gene, Carbon Starved Anther (CSA) shown to yield conditional male sterility in rice. In each case, we will use gene editing, assess the impact on reproductive phasiRNA biogenesis by sequencing, and perform fertility and phenotypic measurements under varied environmental conditions. Ultimately, male-sterile events will be assessed for yield, and suitability in seed production in collaboration with the group of Prof. Graham Moore, with genotyping and phenotyping described in Aim 3.Aim 3. Characterization the alteration and male fertility in the materials from Aim 2.This aim includes the genotyping and phenotyping of the materials including transgenics, edited lines, and TILLING mutants from Aim 2. The work will of course take place in parallel to Aim 2, and as the materials are generated, they will be evaluated. Some materials, such as TILLING lines, are already available, while the transgenics will not be available until later in year 1 and development of those materials is likely to continue into year 2.?
Project Methods
Subaim 1a. Complete the dataset needed to fully characterize wheat reproductive phasiRNAs.The purpose of this subaim is to complete our existing data sets. In our preliminary analyses, we have generated 12 sRNA libraries from hexaploid wheat anthers from two wheat varieties (6 spring and 6 winter, Fielder & Dekan varieties); each variety has 3 replicates from meiotic stage (0.8 to 1 mm) anthers and 3 replicates from pre-meiotic stage (0.5 mm) anthers.Subaim 1b. Genomic analyses to identify phased loci and their miRNA triggers.Next, we'll take the data from subaim 1, and process it through our computational pipelines to identify miR2118 and miR2275 precursors, the miRNAs they encode, as well as the full complement of 21- and 24-nt reproductive phasiRNAs. These pipelines are robust, published,and commonly used in the lab. We would also like to assess sequence similarity among precursors and flanking regions to minimize the number of guide RNAs needed for deletion of these miRNAs in Aim 2.Subaim 2a. CRISPR system and transformation of constructs.The transformation facility at the JIC will be used to implement CRISPR in wheat; this is where the wheat transformation system has been developed and optimised in-house. The JIC team has a wealth of tools available for genome editing, including a wheat codon-optimised Cas9, Cas9 fusions and monocot specific Pol III U6 and U3 promoters from wheat, rice and maize as Golden Gate "MoClo" (Modular Cloning)compatible components. We will transform the spring wheat variety 'Fielder', which is routinely used with an average transformation efficiency of ~20%.Subaim 2b. TILLING as an alternative method to generate genetic variants in wheat DCL5.Wheat has an outstanding and useful TILLING population that is a powerful and inexpensive resource to complement the editing approach described in Subaim 2c. The protein-coding sequences of 1200 and 1535 EMS mutant lines from hexaploid wheat cv. Cadenza and tetraploid wheat (cv. Kronos) respectively have been sequenced using exome-capture. More than 10 million mutations in the sequenced genes were identified and are displayed in a public database.Subaim 2c. Disrupting the biogenesis of 21-nt premeiotic phasiRNAsIn this subaim, we are aiming to phenocopy the P/TMS lines used for two-line hybrid production in rice - this is our desired experimental outcome. In rice, the perturbed loci yield 21-nt phasiRNAs, triggered by miR2118. The only major biogenesis component unique to the 21-mers is the set of miRNA triggers, miR2118. In all other grasses, miR2118 loci are found in two large clusters per diploid genome, corresponding to six clusters in hexaploid wheat, but in our preliminary data, we have identified 13 loci. This includes a substantial cluster of five precursors on chromosome 11.Subaim 2d. Disrupting the biogenesis of 24-nt meiotic phasiRNAsAs described above, in maize, a loss of function of DCL5 (a single copy gene in all monocots examined), results in temperature-dependent male sterility via the complete loss of 24-nt meiotic phasiRNAs. We will determine whether the same loss of function in wheat phenocopies the maize mutant. Deleting DCL5 in wheat is a reasonably straightforward experiment as our preliminary analysis showed it's a single copy gene in each of the three genomes of hexaploid wheat. Other than DCL5, there is only one additional known component specific to and upstream of the production of meiotic 24-nt phasiRNAs, the miRNA trigger (miR2275). In rice and maize, MIR2275 precursors are found in genomic clusters, facilitating their deletion by genome editing, as with miR2118. One outcome of aim 1 will be the identification of the full set of MIR2275 precursors, estimated at two to four loci per homoeologous chromosome set (we found six in our preliminary data); we will design the minimal sgRNA set for their deletion, based on conservation in the precursors (from Aim 1).Subaim 2e. Targeted modification of phasiRNA precursors to yield rice-like P/TGMS loci.This subaim focuses on targeting orthologs of the rice PMS1 and PMS3 mutants. In subaim 1b, we will attempt to identify the wheat orthologs of the rice lncRNA precursors. While that aim is highly risky, if we can find the orthologs, we will use the same CRISPR system to alter the orthologs to mimic the PMS1/3 mutations. Both of the rice P/TGMS loci PMS1 and PMS3 contain SNPs in mRNA precursors of 21-nt premeiotic phasiRNAs. We will design single guide RNAs either delete the entire precursors or replicate the exact mutation observed in the rice lines; in both of those loci, the disruption is in the 2nd or 3rd phasiRNA in each locus.Subaim 2f. The "Carbon-Starved Anther" (CSA) pathway for conditional male sterility.As a final component of Aim 2, we will attempt to replicate in wheat work showing PGMS alleles can be generated by editing in rice of the CSA gene. In rice, mutations in CSA result in conditional male sterility, while short day conditions induce sterility. Editing this gene in rice to create novel PGMS alleles worked extremely well. Thus, as an alternative to altering phasiRNAs (Subaims 2a to 2d), we will develop loss-of-function mutations in wheat CSA orthologs.Subaim 3a. Genotyping of TILLING lines and CRISPR transgenics.After regeneration of transgenic plantlets, altered sequences will be identified and verified by Sanger sequencing of amplicons of the target genes, the conventional method for both TILLING confirmation and CRISPR target genes. Sequencing is performed along with proper wildtype control to exclude any off-target background amplification. SNPs in the flanking sequences are used to separate A/B/D wheat genome homeologs, which is routine in the Moore lab. Sequences are compared to the controls and the reference genome of wheat, which is under continual improvement. Once the mutations are identified, primers will be established for subsequent genotyping to identify homozygous progeny.Subaim 3b. Phenotyping of male fertility in lines genotyped in Subaim 3a.After the mutations are confirmed, male fertility in homozygous plants will be evaluated by pollen staining (see Figure 1C) and visual assessment of anther development, for plants grown under normal conditions (25/20° C, day/night) in the greenhouse. If any of the obtained material has short or pale anthers, or a significant portion of aborted pollen grains, the material is likely male sterile. Replicated progeny from male sterile lines will be regrown under lower temperatures and shorter days to assess whether it is conditional. In addition, male sterile lines will be phenotyped throughout anther development to find the earliest evidence of the primary defect, including using electron microscopy to examine tapetal development.Subaim 3c. Sequencing of small RNAs in mutants to confirm altered phasiRNA biogenesis.For mutants identified as having fertility or anther development defects, staged, isolated anthers will be harvested for small RNA analysis. We will use the temporal map of miR2118, miR2275, 21- and 24-nt phasiRNA accumulation from Aim 1 to selected the most appropriate anther stage for small RNA analysis, linking the target gene with the point at which we anticipate seeing altered small RNA abundances. Two biological replicates will be performed, and data analysed as described in Aim 1, but with an emphasis on the type of alteration anticipated based on the mutant. For example for lines with deletions of subsets of miR2118, we will sample early premeiotic anthers when miR2118 is abundant, and we will sample slightly later when the 21-nt phasiRNAs are abundant, to assess the impact of the partial loss of the trigger miRNA family. In this example, we may be able to associate specific miR2118 family members with specific 21-nt PHAS loci.