Page 265 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

Repeats) gene editing systems, such as CRISPR/Cas9 and CRISPR/Cas12a, are widely used for gene
editing in plants. In 2017, a biotechnology company named Inscripta released the MAD7 nuclease, a
Cas12a-like nuclease enzyme, freely available as a royalty-free tool for both academic and commercial
use, eliminating licensing constraints or royalty payments and enabling users to capitalize on their results.
The novel MAD7 was discovered in the Eubacterium rectale bacteria found in the human gut microbiome
of rural Madagascar resident.

The project utilized the CRISPR/MAD7 gene editing system in the spring wheat cultivar ‘Fielder’, with a
primary focus on drawing conclusions regarding its editing efficiency using different Crispr-RNAs
(crRNAs) with distinct Protospacer Adjacent Motifs (PAM) sequence recognition, targeting the same
gene. MAD7 nuclease recognizes T-rich PAM sequence (YTTN, where Y = T or C, N = A, C, or G).
Additionally, we will explore the impact of Matrix Attachment Regions (MARs, Tobacco RB7 MAR) on the
transgene expression in wheat. MARs are unique regulatory DNA sequences, 100 – 3000 bp AT-rich
fragments, which can bind to the nuclear matrix. The research goal is to mutate the lycopene ε-CYC gene
to redirect the carotenoid pathway towards the accumulation of β-carotene in wheat. We employed the
GoldenBraid cloning system for constructing MAD7 plasmids and used Agrobacterium-mediated
transformation, followed by tissue culture and putative transgenic plants cultivation under controlled
conditions. So far, genomic DNA extraction and PCR confirmed the presence of the MAD7 transgene in
35 out of 57 putative T0 transgenic plants.

Further assessment involved qPCR and Cleaved Amplified Polymorphic Sequence (CAPS) assay to
confirm edits at the target regions. Notably, crRNA (lab# AB341) demonstrated promising results, with 25
out of 30 positive transgenic plants exhibiting edits confirmed by CAPS assay. These samples will be re-
verified by Long-Read Sequencing. The transgene copy number was measured using digital droplet PCR,
with 8 out of 25 having a low transgene copy number (<5). Conversely, qPCR analysis of samples with
crRNA (lab# AB339) did not reveal evident mutations, which was confirmed by Illumina Next Generation
Sequencing. Overall, we aim to use different crRNAs and incorporate specific MARs with the
CRISPR/MAD7 system to enhance gene editing efficiency in wheat.

[P125] IDENTIFICATION OF QTLS FOR PREHARVEST SPROUTING RESISTANCE IN SPRING
WHEAT (TRITICUM AESTIVUM L.). Ramanpreet Ramanpreet , Gurkamal Kaur , Muhammad Iqbal ,
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Curt A. McCartney , Dean Spaner , and Belay T. Ayele . Department of Plant Science, University of
Manitoba, Winnipeg, Canada; and Department of Agricultural, Food and Nutritional Sciences, University
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of Alberta, Edmonton Canada
Correspondence to: gurkamal.kaur@umanitoba.ca

Wheat grain quality is significantly affected by pre-harvest sprouting (PHS), which refers to the
germination of seeds on the mother plant due to the occurrence of wet and humid conditions prior to
harvest. PHS is primarily controlled by seed dormancy, an adaptive trait that blocks the germination of
seeds under optimal environmental conditions. Thus, a low level of seed dormancy leads to increased
susceptibility to PHS. Given that PHS and seed dormancy are polygenic traits governed by multiple
genes, identifying specific genomic regions related to these traits holds significant promise in addressing
the problem of PHS in wheat. Therefore, the objective of this study was to identify QTLs and genetic
markers regulating PHS and seed dormancy through genome-wide association analyses of a mapping
panel that consists of diverse wheat genotypes grown over five different environments. Mature seeds of
the association mapping panel were characterized for their germination index (GI), which exhibited
significant variation in seed dormancy levels among the genotypes. Genotyping of the mapping panel was
performed using a 90k Illumina iSelect SNP array. Mixed linear model (MLM_Q+K) identified 14
significant markers located on chromosomes 4A, 5B and 5D. These significant markers explained
phenotypic variations ranging from 10.22% to 20.82%. A total of three QTLs associated with the 14
significant SNPs were detected on those chromosomes. The loci and markers identified to be associated
with seed dormancy could be used for the development of wheat cultivars with enhanced resistance to
PHS.

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