Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 1530 - Rice | 1080 - Genetics (excludes breeding) | 20% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 1510 - Corn (for sweetcorn use 1480) | 1080 - Genetics (excludes breeding) | 10% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 10% |
| 206 - Basic Plant Biology | 1510 - Corn (for sweetcorn use 1480) | 1080 - Genetics (excludes breeding) | 10% |
| 206 - Basic Plant Biology | 1530 - Rice | 1080 - Genetics (excludes breeding) | 10% |
| 206 - Basic Plant Biology | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 10% |
| 903 - Communication, Education, and Information Delivery | 1510 - Corn (for sweetcorn use 1480) | 1080 - Genetics (excludes breeding) | 10% |
| 903 - Communication, Education, and Information Delivery | 1530 - Rice | 1080 - Genetics (excludes breeding) | 10% |
| 903 - Communication, Education, and Information Delivery | 999 - Citrus, general/other | 1080 - Genetics (excludes breeding) | 10% |
Transposable elements are fragments of DNA that can move from one site in the genome to another and often increase their copy number in the process. The focus of this project is on basic and applied research on transposable elements (TEs), which were first discovered in maize and are now known to comprise the largest component of all sequenced plant (and animal) genomes. The plants studied in this project focus on members of the grass clade, which are the most important source of calories for the human diet. However, because TEs are ubiquitous in all genomes, the findings from this project will inform studies of all crops grown in California, including all fruit and vegetable species. TEs create a significant fraction of genomic diversity, which is responsible for differences in gene expression patterns within a species. For example, they frequently insert into gene regulatory regions where they alter the tissue specific pattern of expression or the activation of transcription in response to stresses such as drought, salinity or cold temperature. In addition, because TEs are themselves induced by a variety of stresses, they are uniquely suited to increase the frequency of mutation when a population is most in need of generating diversity to survive. As mentioned above, TEs comprise the majority of all characterized plant genomes where they generate significant diversity. However, because the vast majority of TEs in all plants and animals are no longer active, it is difficult to determine how a population senses danger and responds by rearranging its genome. It is for this reason that this project focuses on a few closely related rice strains where a TE called mPing is actively amplifying throughout the genome and generating diversity right before our eyes. These studies are important to California because we are particularly interested in understanding how TEs generate diversity that allows plants to adapt to climatic extremes such as temperature, drought and salinity. This proposal also seeks to increase science literacy in the plant sciences. Specifically, we have developed and are expanding the Dynamic Genome courses to involve incoming UCR freshmen in the excitement of authentic research experiences through the analysis of TEs in plant genomes. As such, UCR freshmen will be introduced to the excitement of plant research and obtain the cutting edge tools necessary to continue in research throughout their college career.
For the resequencing effort, one of the four strains will serve as the reference genome for the project and will require an initial build using Illumina 500 bp paired ends, 3 kb and 5kb mate pair libraries and an 8 kb 454 mate pair library to coverage of ~180X. The assembly will be built using SOAPdenovo and/or Velvet software. Given the expected similarity of the 3 other strains, they will be resequenced to ~100 fold coverage using only a 500 bp mate pair library and 2 lanes on the upgraded UCR HiSeq-2000 Illumina machine. SNPs, indels and chromosome rearrangements will be determined for one of the strains, HEG4 that is one of the two parents of the recombinant inbred population that is currently being grown in Arkansas. The other parent is the reference Nipponbare genome. SNPs will be detected using the Broad Institute Genome Analysis Toolkit (GATK). Breakdancer will be used to detect deletions. Detection of smaller indels and other rearrangements will be performed with Pindel and custom Perl scripts.
Target Audience
This project has two major target audiences - undergraduate students who participate in Bio20 and the summer internship experience and citrus researchers. For Bio 20 during AY 2017-18, 13 sections of up to 24 first year undergraduate students participated in an authentic research project that utilized citrus and the citrus genome sequence generated by this project. Furthermore, during summer 2017, 16 rising second year students participated in an intensive summer research project involving the characterization of citrus promoters from genes under circadian (clock) control. In addition, we have made the citrus genome sequence available to a handful of citrus researchers. When the genome sequence is published later this year, that number will certainly increase significantly.
Changes / Problems
In 2016 the direction of this project was changed to the analysis of TEs from the citrus genome. To this end we have spent the past 18 months generating a high quality genome sequence of the Fairchild variety which will be of use to the wider citrus community and will serve as a source of California-relevant projects for our undergraduate students in Bio20. Briefly, progress can be summarized as follows: To build a better reference genome for citrus, we generated 120 fold long reads from Fairchild mandarin using PacBio single-molecule real-time (SMRT) sequencing platform. In total, 3.9 million reads were obtained with an average read length of 9.4 kb. The long reads were assembled using a diploid assembler FALCON and the alternative heterozygous contigs were removed using HaploMerger2. The assembled genome is 360 Mb with contig N50 of 3,635 kb. To further improve the assembly, we generated 150 fold 10x Genomics linked reads and 280 fold bionano genome map. These data were used to scaffold the assembled contigs and fill the sequence gaps between contigs. Finally, we obtained a reference genome of 366 Mb with contig N50 of 10 Mb. All chromosomes were assembled into less than 10 contigs. The assembly is better than reference genomes that were reported in prior studies, such as clementine mandarin (genome size of 301 Mb and contig N50 of 119 kb), sweet orange (genome size of 327 Mb and contig N50 of 50 kb), and Pummelo (genome size of 345 Mb and contig N50 of 2,182 kb). Finally, this project has also involved culturing citrus for the eventual introduction of rice TEs. To this end, culturing of Fairchild is in progress. We restarted the embryogenic callus induction and somatic embryogenesis in the Fall of 2017. We are culturing the embryo like structures both on solid and liquid media. We are optimizing the growth factors for the media so that there won't be any callus browning or cell apoptosis. This experiment will be further standardized using more embryos for reproducibility during coming months. The protocols for callus induction and direct regeneration from cotyledonary explants is being finalized and will be repeated with more explants and can be exploited for doing agrobacterium mediated gene transfer in the Spring 2019. In addition to this, we are developing protocols for embryogenic callus induction using embryos from the developing ovules of Fairchild fruit.
Training & Professional Development
Both the citrus and rice components of this project provided vast training opportunities for both wet and dry (computational) experiences. For first or second year undergraduates: During 2017-2018 thirteen sections of the Biology 20 course analyzed different aspects of the Fairchild genome. In winter 2018 two sections amplified loci associated with resistance to HLB in Fairchild and other varieties in the Citrus Collection. These students were given specific regions of the genome to design PCR primers for amplification and sequencing to identify polymorphisms between the varieties. The other sections of the course identified and amplified putative clock genes from Fairchild. During summer 2018, 17 students (10 first-years and 7 transfers) continued this project to identify clock gene homologs in the Fairchild Mandarin genome. The students started with the protein sequences of the known Arabidopsis clock genes (TOC1, CCA1, PRR7, PRR9, and LHY1) and used TARGeT (target.cyverse.org) to identify candidate genes in Fairchild. The students designed primers to amplify the genes including the putative promoter regions. They analyzed the sequences from other citrus varieties as well. We are planning on submitting these sequences to Genbank. The students cloned the promoter regions each gene with the aim of testing for circadian activity in tobacco leaf assays. After the summer program ended, 12 students enrolled in a research course (Biology 190) to continue the project. During fall 2018 the students moved the cloned sequences into expression vectors and transformed the resulting plasmids into Agrobacterium and infiltrated tobacco leaves. Leaf disks were imaged for three days and activity was detected in the promoter region of one of five tested. The expression was not cyclic. Fourteen new students (all transfer students) in Biology 190 are repeating the fall work with these promoters with a slightly modified experimental design of including the promoter region plus the 5' UTR to see if critical signals were missing from the original cloned sequences. This work was done in collaboration with Dr. Dawn Nagel, an associate professor in Botany and Plant Sciences. For advanced undergraduates: The undergraduate laboratory assistants (ULAs) worked with the students in the sections to help with learning techniques and data analysis. ULAs also receive training in preparing for lab courses and assist with the reagents and supply preparation. The ULAs are a major component of the course and are often the first person the students will ask for assistance. Postdoctoral associate: The citrus genome sequencing project introduced the postdoctoral associate to the field of third-generation sequencing technologies and the computational analysis necessary to perform genome assembly (Pacbio long reads, bionano genome mapping, and 10X Genomics linked reads).
Dissemination Streams
Most of the results have been published or are in preparation. In addition, Wessler receives frequent invitations (about 1/month) to present seminars on the rice project and the Bio20 course.
Next Reporting Steps
Nothing Reported