Page 203 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

examined using the SoyBase database (https://www.soybase.org/) and other bioinformatic tools. Within
the linkage disequilibrium block containing the peak SNP, the GmLAX1 gene was identified at 140.17 kb
upstream of the peak SNP. From the transcriptome data available on SoyBase, this gene has the highest
expression in the root compared to other tissues across developmental stages. To functionally validate
the modulatory role of GmLAX1 in RSA, two guide RNAs within exon 1 were selected for CRISPR/Cas
targeted mutagenesis via Agrobacterium rhizogenes-mediated transformation. Preliminary results
suggest that GmLAX1 is a repressor of secondary roots in terms of their emergence and elongation in
soybean. Thus, the knock-out of GmLAX1 shows potential for enhancing total length and density of the
root system, mainly via its effect on the number of secondary roots. Such root systems may increase the
resilience of soybean cultivars to abiotic and biotic stressors in the face of changing climatic conditions.
These insights may be used to guide breeding strategies aimed at developing soybean varieties with
improved RSA.

[O198] GENE EDITING-ASSISTED FUNCTIONAL GENOMICS STUDIES IN WHEAT (TRITICUM
AESTIVUM L.). Andriy Bilichak, Louie Lopos, Emanpreet Kaur, and Natalia Bykova. Morden Research
and Development Center, Agriculture and Agri-Food Canada, 101 Rte 100 #100, Morden, MB R6M 1Y5,
Canada
Correspondence to: andrii.bilichak@agr.gc.ca

Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation
protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related
genes have been described, the co-delivery of which significantly increases the transformation efficiency.
This study aimed to examine factors affecting Agrobacterium-mediated transformation and gene editing in
wheat (Triticum aestivum L.) to optimize high-throughput gene editing for functional genomics studies.
Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with
GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1)
chimeric gene. We used the Agrobacterium-mediated transformation method and immature wheat
embryos as ex-plants. The T-DNA integrity and transgenes copy number were measured using PCR and
digital droplet PCR. The GE rate was quantified with qPCR and verified with Sanger sequencing.
Eventually, the gRNA activity for 10 different gRNAs targeting 30 loci was correlated with epigenetic
profile (DNA methylation, histone posttranslational modifications, ChIP RNA polymerase, and ATAC-seq)
at the target regions.

Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were
faithfully propagated into the following generation. Both low- and high-copy-number integration events
were recovered in the T0 population with various levels of integrity of the left and right T-DNA borders.
We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome.
A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant
positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary
screening of transgene quality and GE events in the T0 population of plants regenerated through the co-
delivery of GRF–GIF can allow for the propagation of the best candidates for further phenotypic analysis.

[O199] CRISPR/CAS9 BASED LOSS-OF-FUNCTION GENE EDITING CONFERS BROAD-SPECTRUM
2
1,3
1
1
CLUBROOT TOLERANCE IN CANOLA. L. Wang , R. Wen , B. Luo , K. Yang , X. Liu , T.
2
Dumonceaux , G. Peng , and W. Xiao . Department of Biochemistry, Microbiology and Immunology,
1 1
2
2
2
University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5; Saskatoon Research and Development
Centre, Agriculture and Agri-Food Canada, Saskatoon, SK S7N 0X2; and Department of Plant
3
Sciences/Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK,
Canada, S7N 5A8
Correspondence to: wei.xiao@usask.ca; lipu.wang@usask.ca

Canola (Brassica napus L.) is the number one cash crop in Canada and contributes $29.9 billion dollars
to the Canadian economy annually. Nevertheless, the canola industry faces an ongoing threat from
clubroot disease, with an increased number of infested fields each year, especially in Alberta. The causal
agent of clubroot is a protist, Plasmodiophora brassicae Woronin, causing large, disorganized growths
(clubs/galls) on infected roots that disrupt water and nutrient uptake and result in wilting, stunting and
premature ripening of canola. Each large gall contains millions of resting spores persistent in the soil for
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