Location: Bioproducts Research
Project Number: 2030-21410-022-006-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Apr 1, 2024
End Date: Dec 31, 2024
Objective:
Background: Guayule represents an invaluable domestic crop for natural rubber supply security in the U.S., but commercial expansion of this crop is inhibited by poor genomic resources, incomplete definition of the rubber biosynthesis pathway (RBP), and a lack of understanding of how cold temperatures regulate the RBP at the molecular level. In addition, the specific machinery that constitutes the Rubber Transferase (RuT) biosynthetic complex is yet to be identified in plants. The USDA-ARS Bioproducts Research Unit (BRU; Albany, CA) is a partner in a Joint Genome Institute (JGI)/DOE Community Science Project (CSP), Molecular basis of natural rubber biosynthesis in Parthenium argentatum (Guayule) (ARS-LBNL Agreement 58-0310-2-001N). The Cooperator serves as the project’s Principal Investigator, and is working with BRU to analyze sequence data produced by the JGI. To date more than 10 terabytes of sequencing data, both long (PacBio) and short (Illumina) read, as well as genome and transcriptome, have been generated. These data are being used to assemble and annotate the genomes for three guayule accessions - a diploid line CAL3, a tetraploid AZ6 and hybrid AZ2. In addition, JGI has sequenced transcriptome data from a multisite field trial where tissue was collected from all three accessions at multiple timepoints before and after cold-induction of the rubber transferase. A chromosome-level phased diploid assembly for the diploid (CAL3) and hybrid AZ2 is complete, and the assembly for tetraploid AZ6 is in process.
Objective: We propose a collaborative effort to analyze the genomic sequences generated by this Community Science Project, aiming to 1) create high-quality guayule reference genomes representing the three focal germplasm, and 2) identify regulatory elements dictating how cold affects guayule rubber biosynthesis. The sequence data at the heart of this proposed work are available to CSP members only for a limited time, during which members will analyze the data and prepare associated publications.
Approach:
Approaches:
1. Creating high-quality guayule reference genomes:
A. We will evaluate the quality of the CAL3 and AZ2 genome assemblies using multiple approaches. Evaluation methods include mapping DNA reads against assemblies, BUSCO analysis, whole genome alignment, collinearity with genetic maps, LTR assembly index, and k-mer spectrum analysis. Similar analyses will be applied to AZ6 as it becomes available.
B. Annotate genetic assemblies, analyze pseudo genes, and assign putative gene function through sequence similarity to known genes from model organisms (Ontology assignment).
C. Use prior knowledge of candidate genes within the rubber biosynthesis pathway to examine the evolution of the RBP within guayule – with a specific focus on expansions and duplications within the rubber transferase components that would be specific to guayule versus other plants in the Asteraceae.
2. Identify regulatory elements dictating how cold affects guayule rubber biosynthesis:
A. Create transcriptional profiles of the multi-site field-generated RNA-seq data. Identify genes that are differentially abundant between tissues (old and young stem) as well as temperature (before and after induction of the RBP) between the three accessions.
B. Develop a co-expression network to identify genes that are expressed in a similar manner in response to temperature and correlate these modules of genes to rubber content and enzyme activity. Use these modules of highly trait-expression correlated genes to identify transcription factors that might be regulating those genes. Then use in silico approaches to identify transcription factor binding sites that can be tested in vitro. From these data, select a few TF or trait-associated genes that would be targets for functional evaluation.
Ultimately, the information developed will facilitate mechanistic studies of the regulatory control of rubber biosynthesis in plants, guayule crop improvement, and potentially, the introduction of rubber biosynthesis pathways in alternative systems such as tobacco.
