Marker development and establishment of qPCR-based screening for verticillium stripe disease in Canola
Term: 4 years, beginning 2024
Status: Ongoing
Researcher(s): Harmeet Singh Chawla, U of M
SaskCanola Investment: $129,375
Total Project Cost:
Funding Partners: ADF, WGRF, MCGA
Objective
We will screen a diverse collection of 250 Brassica napus genotypes (along with appropriate controls) for verticillium stripe resistance using accurate quantitative PCR (qPCR).
Genome-wide association analyses (GWAS) will be used to identify verticillium stripe (VS) resistance QTLs. Although there have been a few recently funded projects aimed at mapping VS resistance in canola; we plan to use qPCR-based phenotyping (from Objective 1) rather than visual scoring which should give a precise identification of Verticillium longisporum infection and separate it from blackleg (visually similar).
Once we have identified the genetic polymorphisms underlying resistance QTL, we would develop KASP markers. These KASP markers would be instrumental in introgressing newly identified verticillium stripe resistance alleles into elite Western Canadian breeding material and cultivars.
Project Description
Verticillium longisporum is a soil-borne fungal pathogen that can infect a range of host plants, such as horseradish, canola, cabbage, and cauliflower. In canola, the fungus enters the plant through the roots and colonizes the vascular system, causing verticillium stripe (VS) disease. However, phenotyping for VS resistance is not an easy process as V. longisporum does not cause conventional wilting symptoms in canola. Rather, the crop ripens prematurely, making disease symptoms difficult to distinguish from natural senescence until after harvest. This lack of clear symptoms leads to high degree of variation in VS resistance screening thereby complicating the process of identification of genomic regions controlling the disease resistance.
Through this proposal, we aim to facilitate the phenotyping process by developing a qPCR (quantitative polymerase chain reaction) based method for quantification of V. longisporum levels in canola under field conditions. V. longisporum infiltrates the host plant through its roots; however, the fungus encounters impediments in reaching the shoots when infecting resistance genotypes. The barriers formed in the hypocotyl tissue upon infection, both morphologically and biochemically, effectively hinder the pathogen's spread to the aerial parts of the plant. Thus, it can be postulated that a resistant B. napus plant would exhibit a reduced presence of fungal DNA in its hypocotyl compared to a susceptible genotype. This disparity in fungal DNA content between resistant and susceptible plants can be leveraged to characterize the resistance of various canola genotypes against V. longisporum. Therefore, we propose to perform a genome wide association study (GWAS) using the qPCR screening method on approximately 250 diverse B. napus genotypes to identify stable VS resistant quantitative trait loci (QTL). The GWAS panel would comprise commercial canola cultivars, re-synthesized B. napus genotypes from Dr. Isobel Parkin and other genetically diverse germplasm from Dr. Sally Vail.
Compared to visual scoring, qPCR has several advantages:
1. Quantitative measurement: Visual scoring is a qualitative measurement technique that relies on subjective visual assessments of disease symptoms, such as wilting or yellowing of leaves. While visual scoring can provide useful information about the severity of disease symptoms, it does not provide a quantitative measurement of the pathogen load in canola plant. In contrast, qPCR provides a precise quantitative measurement of the amount of pathogen DNA present in the sample. The ability to quantitatively measure the amount of pathogen DNA in a sample is especially important under field conditions, where the pathogen load may be low or where multiple pathogens are present. In such cases, visual scoring may not be able to accurately distinguish between different pathogens or precisely assess disease severity, whereas qPCR can provide a more accurate and precise measurement of pathogen load.
2. Specificity: Visual scoring may not be able to easily differentiate between different fungal pathogens (such as Leptosphaeria maculans) or other factors (e.g., senescence) that may be causing disease-like symptoms. Additionally, qPCR can also distinguish between different species of Verticillium such as V. longisporum and V. dahliae.
3. Reproducibility: qPCR is a highly reproducible technique, with low variability between replicate assays, whereas visual scoring can be subjective and may vary between different observers who are doing surveys for each province.
Following the identification of VS resistance polymorphisms, we aim to develop KASP markers specific to the most important loci. These KASP markers will be used in the University of Manitoba B. napus breeding program and AAFC canola breeding programs for marker-assisted selection.