Performing Department
Plant and Microbial Biology
Non Technical Summary
With support from the NIFA Fellowship, I expect that integration of rice chromatin biology (Kim), cell isolation technique (Dr. Okamoto) and genetics and seed development biology (Dr. Fischer), will create a highly synergistic and well-coordinated team for investigating gametophyte development. The proposed research will provide novel insights into the epigenetic programming of pollen development, which will provide valuable resources for developing new technologies that increase crop yield to feed growing populations and address the problem of world hunger.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
(N/A)
Developmental
20%
Goals / Objectives
To evaluate the successful performance of the proposed research, a list of milestones follows: 1) Have ~3,800 vegetative nuclei, sperm cells, and pollen grains from wild-type and ~3,800 sperm cells and pollen grains from ros1a plant collected. About 3,300 sperm, vegetative nuclei, and pollen grains from wild-type; and sperm cells and pollen grains from ros1a mutants are isolated (~1,500 cells/pollen for DNA methylation mapping, two biological replicates of ~150 cells/pollen for RNA mapping, and two biological replicates of ~1,000 cells/pollen for small RNA mapping). With help of Dr. Okamoto and his technicians, I obtain enough materials by a manual cell isolation method in one month. 2) Have Illumina sequencing libraries constructed. DNA and RNA are purified from the collected cells, and BS-seq and RNA/small RNA-seq libraries are constructed. The libraries are sequenced using the Illumina HiSeq2000 platform in Vincent J. Coates Genomic Sequencing Laboratory in UC Berkeley. 3) Have hypotheses answered through computational analyses of genomic data sets. Sequenced reads from BS-seq, RNA-seq and small RNA-seq are aligned against the reference rice genome. I identify 1) If vegetative and sperm cells have differentially methylated elements; 2) If these differentially methylated elements are also differentially expressed in those cells; and 3) If small RNA population is observed in ROS1a inactive cells. 4) Have data published in a well-respected scientific journal. The results from the computation analyses are organized in a manuscript for publication.
Project Methods
AIM 1. Identify ROS1a-mediated DNA demethylation in pollen. 1A) Isolate vegetative nuclei, sperm cells, and pollen in rice wild-type and ros1a mutants. Experimental method. The anthers will be collected from flowers before dehiscence. Pollen grains will be collected in sucrose solution by gently crushing the anthers as shown previously. For sperm and vegetative nuclei isolation, anthers will be crushed in mannitol solution to burst the pollen. In the mannitol solution, two sperm cells and the vegetative nucleus are released through three apertures of a pollen grain. The sperm and vegetative nuclei will be collected manually with a microinjecter under bright field and fluorescence microscopy. The collected materials will be thoroughly washed in clean mannitol solution before freezing in liquid nitrogen for storage. 1B) Generate a map of DNA methylation in wild-type and ros1a mutants. Experimental method. I will construct BS-seq libraries of DNA extracted from isolated sperm cells, vegetative nuclei, or pollen grains and will elucidate their DNA methylation profiles. About 1,000-2,000 cells or pollen grains, containing roughly 0.5-1 ng of genomic DNA, will be lysed at 98 oC for 10 minutes. After lysis, I will shear the DNA to 150-1,500 bp range, then clean the DNA using AMPure XP beads (Beckman Coulter). The sheared DNA will be used to generate libraries using Ovation Ultralow Methyl-Seq Library System kit (NuGen). A total of five BS-seq libraries will be sequenced on the Illumina HiSeq2000 platform in one 100bp sequencing lane. Each sample will have >10x genome coverage. AIM 2. Determine if DNA demethylated elements are activated and produce small RNAs. 2A) Produce transcriptomes in sperm and vegetative cells from wild-type and ros1a plants. Experimental method. From the isolated sperm cells, vegetative nuclei and pollen grains from Aim 1A, total RNA will be extracted using the RNAqueous-Micro Kit (Ambion). cDNA will be synthesized and amplified using the Ovation RNA-seq FFPE system kit (NuGen), as previously described using 5 ng of total RNA samples. Ovation-amplified cDNA will be used to construct an Illumina library by Encore Rapid Library System kit (Nugen). A total of ten (including one biological replicate) RNA-seq libraries will be sequenced on the Illumina HiSeq2000 platform in one 100bp sequencing lane. Each sample will have >10x cDNA coverage. Alternative approach. It has been shown that nuclear and cytoplasmic RNA populations are similar in human cells. Thus, the above method is likely to produce comprehensive maps of RNA in vegetative and sperm cells. However, we cannot eliminate the possibility of excluding mRNA predominantly found in the vegetative cytoplasm. To complement the proposed method, I will extract RNA from pollen and sperm cells, which will be isolated using Percoll gradient centrifugation as described previously. FACS is not suitable for isolating cells for RNA experiments, because sperm cells are found inside a vegetative cell so FACS separated sperm isolate is likely to be contaminated with cytosolic RNA from the vegetative cell. Through the computational analysis, I will be able to obtain the vegetative cells' transcriptional landscape indirectly by subtracting the sperm transcriptome from the pollen transcriptome. In addition, with the larger amount of RNA (~500ng) from the cells isolated via Percoll gradient centrifugation I will construct RNA-seq libraries of samples with depleted ribosomal RNA using the Ribo-Zero kit (Epicentre) instead of isolating mRNA using oligo-dt probes. This way, I will be able to obtain RNA data with a full representation of non poly-adenylated RNAs such as non-coding RNAs, unlike the proposed method in Aim 2A that predominantly identifies the expression level of poly-adenylated mRNA. By performing both RNA extraction methods, I will be able to obtain near-complete landscapes of sperm- and vegetative cell-specific transcriptomes. 2B) Measure small RNA populations in sperm cells and vegetative nuclei isolated from wild-type and ros1a pollen. Experimental method. To obtain a profile of the nuclear small RNA population of wild-type and ros1a sperm and vegetative cell, I will construct small RNA libraries of sperm cells, vegetative cells or pollen grains isolated from Aim 1A, using the TruSeq small RNA preparation kit (Illumina). A total of ten (including one biological replicate) small RNA-seq libraries will be sequenced on the Illumina HiSeq2000 platform in one 50bp sequencing lane. Each sample will have >10x small RNA genome coverage.