Page 174 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

and develop genomic selection (GS) models for improving leaf rust resistance in winter wheat. The study
involved a GWAS panel comprising around 300 western Canadian winter wheat breeding lines and
cultivars, and 100 winter wheat breeding lines and cultivars from the USA, eastern Canada, and Europe.
The GWAS panel was assessed for leaf rust resistance in seedling tests, with multiple P. triticina races. In
addition, this panel was tested for resistance in inoculated field trials in Winnipeg and Morden, Manitoba,
following randomized alpha lattice designs, with two replicates per field trial during the 2022-23 and 2023-
24 growing seasons. The genotyping was done using the Illumina Infinium Wheat Barley 40K SNP array
and the 25K wheat Infinium array. The DNA marker and leaf rust datasets were used for GWAS. Here,
individual markers were tested for association with leaf rust reactions (field and seedlings). Significant
QTLs associated with leaf rust resistance have been identified from the GWAS analyses, indicating the
presence of Lr genes in the GWAS panel. However, the specific identity of these genes has yet to be
determined. The leaf rust and SNP marker data were also used to develop genomic selection (GS)
models for estimating leaf rust resistance in CWRW breeding germplasm. The accuracy of these genomic
selection (GS) models for predicting the leaf rust resistance of wheat germplasm was evaluated through
cross-validation. Improved understanding of the resistance genes in Canadian winter wheat and DNA
markers for selecting these genes will improve the efficiency of wheat breeding programs. Improvements
in breeding efficiency will accelerate genetic gain in wheat breeding programs and result in the
development of new improved varieties for cultivation in Canada.

*[O139] UNVEILING A DNA VIRUS SECRETS: DE NOVO METHYLATION PROFILING OF
GRAPEVINE RED BLOTCH VIRUS VIA LONG-READ SEQUENCING. Vahid J Javaran , Pierre
1,2
1
2
1
1
Lemoyne , Dong Xu , Dave T Ste-Croix , Peter Moffett , and Mamadou L Fall . Saint-Jean-sur-Richelieu
1 1
Research and Development Centre, Agriculture and Agri-Food Canada, 430 Boulevard Gouin, Saint-
Jean-sur-Richelieu, QC J3B 3E6, Canada; and Centre SÈVE, Département de Biologie, Université de
2
Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, QC J1K 2R1, Canada
Correspondence to: vahid.jalalijavaran@agr.gc.ca; mamadoulamine.fall@agr.gc.ca

Geminiviruses are a diverse group of single-stranded DNA (ssDNA) viruses known for their significant
impact on agricultural crops globally, including the grapevine industry. Grapevine red blotch virus
(GRBV), a member of the Geminiviridae family specifically classified under the genus Grablovirus, is the
causal agent of Grapevine Red Blotch Disease (GRBD). GRBV causes severe economic losses in the
grape and wine production sectors by affecting the quality and quantity of grape fruits. GRBV has a
monopartite circular ssDNA genome and is transmitted by the three-cornered alfalfa hopper. There have
been no successful reports on the enrichment and isolation of viral particles from GRBV-infected plants,
which hinders the development of rapid and field-deployable serological tests that rely on the virion.
Consequently, we hypothesized that the coat protein (CP) region of the GRBV genome is highly
methylated, resulting in compromised translation and synthesis of the CP and the formation of the virion.
Therefore, we used a novel methodology for de novo methylation profiling of GRBV, leveraging the
capabilities of Nanopore sequencing to analyze the virus in its native circular form. Our comprehensive
approach involved the extraction of DNA from three distinct GRBV-infected grapevine samples that
display red blotch symptoms, and a PCR product of GRBV as a negative control. The protocol entailed
several enzymatic treatments to linearize the unwanted circular dsDNA, followed by exonuclease
treatment to remove all linear DNAs. A subsequent sequencing step, combined with the super accurate
Dorado's modified base calling model, enabled the identification of 5mC and 6mA methylations on GRBV
genome and in different coding regions. Our findings revealed a remarkable methylation pattern within the
GRBV genome, particularly within the CP coding region which exhibited significantly elevated levels of
5mC methylation compared to other viral genes. This unexpected hypermethylation suggests a pivotal
role of the CP gene in the GRBV-grapevine interaction during the infection lifecycle and could provide
insights into the mechanisms of viral gene regulation and virion stability. Our research provides a valuable
foundation for future electron microscopy-based investigations into the role of 5mC methylation in the viral
CP gene and its impact on virion formation and integrity, offering novel insights into virus-host interactions
and the epigenetic mechanisms influencing viral pathogenicity.

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