Location: Crop Germplasm Research
Project Number: 3091-21000-044-005-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Jan 1, 2021
End Date: Dec 31, 2021
Objective:
The objective of this research is to develop an effective genetic system for increased cotton production under heat stress environments. A major threat to cotton production is abiotic stress due to climate changes. Global warming and consequent heat waves are detrimental to the photosynthesis of which chemical byproducts can damage the machinery of converting sunlight into sugar of the cotton plant. This project explores new methods and resources to tackle the issue for heat tolerance and the increased productivity of fiber and cottonseed in cotton hybrids that may capture desirable heterosis.
Approach:
This research project involves a series of experiments to be performed at USDA Agricultural Research Service. Upon the regeneration of Coker 312 cotton transformants containing the recently engineered cotton D1 gene, we will propagate the T1 seed in USDA greenhouse in College Station, TX. The genomic DNA from individual T1 cotton plants will be extracted for lab evaluation with PCR and sequencing experiments. T2 seed from the positive T1 plant will be developed for backcrossing to the elite cotton cultivar(s) in the greenhouse. The backcrossed cotton plants will be subsequently advanced for further lab evaluation, heat stress test, and eventual yield trial. We will also characterize molecularly identified meiotic phasiRNA genes and other candidate genes with classically identified nuclear male sterility genes. Sequence homology and functional annotation will be analyzed in Upland cotton genome for dicer-like (DCL) loci that specifically regulate meiotic phasiRNAs in cotton anthers. At least three candidate genes will be selected to design the double guide RNAs for target gene knockout in elite Upland cottons using the CRISPR/Cas9 technology. Alternatively, we will use RNAi technology to knockout the function of these target gene transcripts. A multi-target vector will be used to construct gene-editing assembly. The constructs will be validated by transient assays, and the positive plasmids will be transformed into the selected cotton accessions in collaboration with the collaborator. The transformed cottons will be evaluated for CRISPR/Cas9-induced mutagenesis with any changes of targeted DNA sequences and phenotypic observations. The resulting cotton mutants will be regenerated and evaluated for reproductivity of aborted pollen gametes and for propagation of pure male sterile seeds. They will be used as female parents to be crossed with the elite male parents that have normal wild-type fertility and complementary agronomic performance to evaluate the heterosis of F1 cotton plants.
