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CRISPR/Cas9 for crop improvement

Since their functional characterization in 2012, CRISPR/Cas proteins have quickly become a valuable tool for basic and applied research. We are using codon-optimized Cas9 proteins to induce specific modifications in wheat and potato genes. This allows us both to characterize gene function and to induce specific variants that confer novel traits with agronomic and nutritional benefits. As we learn more about how specific genetic variants affect agricultural traits of interest, CRISPR/Cas represents a powerful tool to efficiently and predictably combine beneficial alleles to complement traditional crop breeding methods.

Wheat

The goal of our project is to characterize and integrate beneficial genetic variation into winter wheat varieties of economic importance for Colorado growers. We have identified elite breeding lines from the CSU wheat breeding program that are amenable to transformation and perform well in tissue culture. We are transforming callus tissue from these lines with multiplexed CRISPR/Cas constructs using biolistics and Agrobacterium methods. Our target traits include grain yield and quality, biotic and abiotic stress resistance and herbicide tolerance.

 

Potato

Colorado’s San Luis Valley is the second largest fresh potato growing region in the U.S. In collaboration with the San Luis Valley Research Center, we are applying CRISPR/Cas to generate gene knock-outs and perform base editing in elite Russet and Yellow potato varieties. Starting with internode and petiole tissues, we are inducing callus formation and transforming with CRISPR/Cas constructs targeting genes involved in tuber quality and abiotic stress resistance.

Industrial Hemp

Recent clarifications regarding regulation of cannabis use together with a global expansion of the CBD market resulted in an explosion in demand for industrial hemp production in Colorado. As regulated hemp cultivation in Colorado exponentially increased since 2014, issues related to poor seed genetics arose. Generating improved hemp varieties adapted to Colorado and compliant with Farm bill’s restrictions is key to the successful adoption by Colorado growers. Together with the current expansion of hemp breeding efforts, genome editing has the potential to generate hemp varieties with controlled alkaloid production and improved agronomic performances.

We are testing several hemp varieties grown in Colorado for their amenability to tissue culture and transformation protocols and we will exploit the CRIPSR-Cas9 technology to generate novel hemp varieties.

This research is part of the “Solutions to Crop Commodity Challenges” program at CSU and is supported by Nutrien and CSU’s Office of Vice President for Research (OVPR). Contact: Dr. Karl Ravet (karl.ravet@colostate.edu)

TILLING

We are using chemically-mutagenized TILLING populations to characterize genes involved in grain quality, production and disease resistance traits. Wheat has a polyploid genome, so most target genes are present in two or three copies. We are combining mutations in each copy using custom-designed KASP and CAPS molecular markers to develop plants carrying null mutations in every copy. Once homozygous materials are developed, we analyze the phenotype of mutant lines to characterize gene function and to assess their potential to deliver improved traits in elite wheat varieties.

Wheat spikelet number

There is an urgent need to improve wheat yields to meet the food demands of a growing world population. Among the many components contributing to overall yield, spikelet number is an important determinant of grain number. We are performing field-based genetic mapping studies to identify the genetic variant underlying a QTL for spikelet number using a winter wheat mapping population. We are using exome sequencing to generate high-resolution markers, RNA-seq to identify candidate genes and genomic databases to characterize natural genetic variation. An improved understanding of the genetic variation associated with yield components will enhance our ability to breed for higher-yielding wheat varieties.

This project is funded by USDA-NIFA and IWYP as part of the Wheat Coordinated Agricultural Project (WCAP).

Virus resistance

Wheat Streak Mosaic Virus resistance

Wheat streak mosaic virus (WSMV) causes yield losses in cereal crops in many parts of the world, but is particularly prevalent in the Great Plains of the U.S. Transmitted by the wheat curl mite, there are no effective chemical controls and only limited sources of host genetic resistance. The WSM2 locus is one such source identified in a Colorado breeding line (Tan et al. 2017). We are using transcriptomics and comparative genomics to characterize the WSM2 locus to shed light on the virus resistance mechanism. This will help design more targeted approaches to identify and engineer novel genetic variation to counter this important disease.

Cas13a-mediated virus resistance

To date, most applications of CRISPR in plants have involved the use of Cas9 and its variants to engineer changes in the plant’s own DNA. The discovery of different classes of Cas enzymes open the door for more diverse applications. Cas13a targets single stranded RNA sequences and proof-of-concept studies in several plant species demonstrate its potential to degrade single stranded RNA viruses. We are developing Cas13a-expressing wheat plants to target WSMV and other viral pathogens to assess the potential of this technology to confer durable viral resistance in wheat.

Ambient temperature

To maximize their reproductive success, plants respond to environmental fluctuations so that critical stages of their development coincide with optimal conditions. In wheat, the genetic pathways regulating the vernalization and photoperiod responses are well characterized, but we know very little about how plants regulate their development in response to fluctuations in ambient temperature during the growing season. We are applying bioinformatics and modeling approaches to analyze large volumes of environmental and breeding data to dissect the ambient temperature response and to characterize genetic variation underlying the response. An improved understanding of these pathways will aid the development of varieties more resilient to rising global temperatures.

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