2013 Annual Report
1a.Objectives (from AD-416):
The objective of this SCA is to increase our understanding of plant genomes through sequence generation and characterization.
1b.Approach (from AD-416):
Plant genomes are large and complex genomes, making them a challenge to sequence and characterize their genomes’. With recent changes in sequencing technology it is now possible to generate sequence at fraction of the cost. But the sequence that is generated has different qualities that will require changes in the way we process and interpret this sequence. Experimental and computational approaches will be reviewed and develop to make use of the new short read technologies. This will include library development, integration of different sequencing methodologies, and development of computational pipelines to process, store and interpret the data sets.
In FY 2013 this collaboration focused on the development of resources to facilitate the assembly of plant genomes with a view towards understanding the functions of gene networks. For this work, the group uses our technology to produce genome sequence to identify variation in the genome’s sequence, gene expression, and methylation-state and their relationship to plant development and the expression of agronomically important traits. As part of this collaboration, members have monthly project meetings, where we discuss the production and analysis of sequence, technology transfer between groups for library production as well as other downstream issues. In the last year we have experienced moderate success with the PacBio Systems technologies combined with short read sequencing from the more established Illumina technology for error correction. The project team has also discussed recent emerging methodology to assemble the sequence, and has tested combining these approaches. In both sequencing and assembly the integration of multiple approaches improved the length and accuracy of the genome sequences over what would have been achieve with either technology alone. Collaborators have worked with personnel in each other’s groups to optimize sequence and assembly approaches using rice as a control. This work was in collaboration with the NSF-funded wheat gene-space project. In addition, this collaboration has supported the sequencing and analysis of small RNA libraries, cDNA-based libraries (RNA sequence), and genomic libraries from several plant species. In the last year, the focus has been on rice, maize, wheat, and sorghum. This data has supported the baseline annotations and regulator sequence objectives of the in-house project. We continue to focus on integration of methylation and transcription profiling. With respect to the maize flower development objective, analysis workflows were updated, analyses of co-expressed genes were further refined, and new work involving the development of improved methods to identify the portion of the DNA sequence a transcription factor will bind was begun. In some cases the binding of DNA by transcriptions factors will promote or inhibit the expression of genes in the surrounding region. This work provides insights into genes that contribute indirectly to crop yield. We continue to refine our computational pipeline to monitor the methylation status of the genome, using multiple approaches. The first approach measures, indirectly, the methylation of status of individual sites on the genome. The second approach monitors the small RNA molecules that provide substrates to support changing the methylations status of the genome. By profiling the methylation status of the genome, it is possible to characterize context-specific shifts in methylation on a genome-wide scale across different tissues and developmental stages in maize. The work supports genetic and molecular characterization of gene networks known to be associated with a plants development (flower, stem and root) and its response to the environment. In the past year, additional gene promoters have been screened and we have identified genes that control the expression of genes that regulate root and flower development, as well as genes associated with drought and/or heavy metal stress. We have further developed the genetic resources for analyzing genes found to be “hubs”, i.e., highly connected nodes in the gene network. Using these genetic resources we have begun the validation of the genes binding DNA in plants, by profiling their expression in plants which do not normally contain these genes. Of the 28 putative transcription factors tested, 35% show repression, 35% activation and 30% show no change in expression in these plants. We developed a second approach, that is able to detect the binding of protein to DNA, and applied it to four test cases. Each of these demonstrated repressor activity. We screened 60 gene mutants for short root phenotype. Two mutants showed a short root phenotype.