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Identification of expression QTLs (eQTLs) for Fusarium head blight resistance and susceptibility in wheat

Principal Investigator: Therese Ouellet  

Research Institution: Agriculture and Agri-Food Canada

Timeline: April 2014 – March 2018  

Objectives:

  • Identify genetic markers associated with gene expression patterns that correlate with resistance or susceptibility to Fusarium head blight (FHB) in spring wheat.
    • Determine the most suitable technology for high throughput expression analysis of wheat samples. Determine the most suitable technology for large scale expression profiling experiments of a wheat double haploid (DH) population of 85 individuals to identify candidate differentially expressed genes associated with resistance (R) or susceptibility (S) to FHB. This step will include the testing of parents from two well-characterized spring wheat populations, Wuhan x NuyBai and HC374 x Shaw, segregating for FHB resistance, to validate the choice of technology.
    • Identify expression QTLs (eQTLs) for FHB resistance and/or susceptibility. Determine the global expression profiles of the 85 DH lines from the cross Wuhan x NuyBai.
  • Identify additional wheat breeding material carrying the eQTLs discovered in Objective 1.
    • Assemble and characterize a panel of spring wheat germplasm segregating for FHB resistance. Obtain a collection (panel) of about 120 wheat breeding lines/cultivars from Eastern and Western Canadian breeding programs, with lines contrasting for resistance/susceptibility to FHB from each program. Perform a comparative field evaluation (3 years of field experiment) of the panel of lines for FHB disease resistance to obtain a relative ranking between all the lines in the panel. Test the genetic markers (associated with eQTLs identified in objective 1) on the panel of lines. Correlate the marker data with the FHB resistance phenotype for the panel of lines.

Impacts:

  • Identification of more specific markers for known QTLs or of novel QTLs for FHB resistance, and of germplasm carrying those novel markers/QTLs can complement the present FHB resistance breeding efforts by providing additional options for the design of breeding strategies.
    • An eQTL and a corresponding gene have been shown to be located within the interval for the 2DL QTL for FHB resistance. The gene is possibly directly contributing to the QTL effect and development of genetic markers for it will improve on markers currently in use for the 2DL QTL, making the QTL easier to track in breeding material.
    • One of the genes closely associated with the 2DL QTL is not part of the genes identified in the wheat genome annotation RefSeqV1.0 released in early 2017. The full sequence of that gene, temporarily call UN25696, has been defined and the information recently submitted to the wheat genome annotation team in time for inclusion in the next RefSeq version. This contributes to the polishing of the complete list of genes identified in the wheat genome.
    • The list of candidate genes from the wheat 2DL chromosome to be tested for the presence of an eQTL was established from collaborative work with an Italian group who did high throughput RNA sequencing (RNASeq) of a pair of sister lines containing either the resistant or susceptible allele for the 2DL QTL for FHB resistance. That work was recently published in an international scientific journal with broad readership (Biselli et al). It is the foundation for the identification of eQTLs for the 2DL QTL that was done in this project.

Scientific Summary:

Even though a majority of wheat breeding programs in Canada have deployed significant efforts towards improving resistance for Fusarium head blight (FHB) during the last 20 years, progress has been slow, and FHB still remains a priority problem for the industry. The identification of genetic markers associated with resistance to FHB has accelerated progress by helping breeding programs for spring and winter wheat to track genetic loci for quantitative traits (QTLs) contributing to that resistance. QTLs for resistance to FHB have been identified so far from field disease ratings and DON accumulation data. However, the QTLs identified so far explain only part of the FHB resistance observed in the resistant germplasm, and the genes responsible for the QTL effects and their mechanisms of action are not well understood. Only the gene responsible for the FHB1 resistance has been identified, and its function is still to be understood.

This project used a new approach complementary to QTL mapping, differential gene expression, to identify genetic markers associated with expression QTLs (eQTLs) that directly contribute to the known QTLs. Additional QTLs not detected by conventional marker discovery work using field data were also identified. Identification of more specific markers for known QTLs or of novel QTLs for FHB resistance, and of germplasm carrying those novel markers/QTLs will complement the present FHB resistance breeding efforts by providing additional options for the design of breeding strategies. The project has made significant progress.

Objective 1:

Novel bioinformatics methods to analyse RNASeq data for the identification of eQTLs were developed in collaboration with the group of Y. Pan (NRC Ottawa). These are now being applied to identify eQTLs associated with the 5AS QTL for FHB resistance in the double haploid population from Wuhan1 x Nyubai. A list of candidate genes possibly contributing to the QTL was developed. Access to a fine mapping population for the 5AS QTL will be required to reduce the number of candidate genes. In addition, the 5AS QTL was shown to be strongly correlated to the level of Fusarium in the first few days after inoculation.

A separate collaboration with the group of G. Valè (Italy) to produce and analyse additional RNASeq data has led to the identification of an eQTL within the interval for the 2DL QTL for FHB resistance, and of the corresponding gene. A similar result was obtained from a second mapping population derived from the same genetic material. Additional material from other breeding programs is being tested to further support the role of the identified candidate gene. Development of genetic markers has been initiated to distinguish the resistant and susceptible alleles of that gene.

Objective 2:

This objective was completed. A panel of 120 wheat lines segregating for FHB resistance and contributed to by 6 Canadian wheat breeding programs was assembled and comparative field characterization performed for a third year by the team of S. Khanizadeh. This included disease rating data and DON analysis. DNA was been extracted from all the lines in the panel and will be used for genotyping using the genetic markers in development for the 2DL candidate gene identified under Objective 1. It is hoped to identify additional germplasm carrying that eQTL on 2DL.

Next Steps:

The identification of a gene with an eQTL within the interval for the 2DL QTL for FHB resistance has raised the interest of other wheat breeding programs, including those in Canada, at CYMMIT, Mexico, and at USDA, USA. Those groups have the 2DL QTL present in their germplasm and breeding material; however, the genetic markers currently in use in their respective programs are not optimal. It is hoped that genetic markers associated with our candidate gene will be more specific, and possibly identify a gene directly contributing to the phenotype of the 2DL QTL. Collaborations have been established and breeding material from those breeding programs is now included in our assays.

RNASeq analysis of samples from the double haploid population has also identified candidate genes for the 5AS QTL for FHB resistance; however, it is too large of a list to identify a testable number of candidate genes. International collaborations are being pursued to keep narrowing in on solutions.

External Funding Partners:

This research activity was part of the National Wheat Improvement Program Cluster led by the Western Grains Research Foundation (WGRF).

Funding for this project was provided in part by Agriculture and Agri-Food Canada through the Growing Forward 2 (GF2) AgriInnovation Program and in part by Canadian Field Crop Research Alliance (CFCRA) members. Grain Farmers of Ontario is a founding member of the CFCRA.

Project Related Publications:

Biselli, C., Bagnaresi, P., Faccioli, P., Hu, X., Balcerzak, M., Mattera, M.G., Yan, Z., Ouellet, T., Cattivelli, L., Valè, G. 2018. Comparative transcriptome profiles of near-isogenic hexaploid wheat lines differing for effective alleles at the 2DL FHB resistance QTL. Frontiers in Plant Science. 9: 37.

Fauteux, F., Wang, Y., Rocheleau, H., Liu, Z., Pan, Y., Fedak, G., McCartney, C., and Ouellet, T. 2019. Characterization of QTL and eQTL controlling early Fusarium graminearum infection and deoxynivalenol levels in a Wuhan 1 x Nyubai doubled haploid wheat population. BMC Plant Biology. 19. 10.1186/s12870-019-2149-4.

Hu, X.K., Rocheleau, H., McCartney, C., Biselli, C., Bagnaresi, P., Fedak, G., Yan, Z., Valè, G., Khanizadeh, S., and Ouellet, T. 2019. Identification and mapping of expressed genes associated with the 2DL QTL for Fusarium head blight resistance in the wheat line Wuhan 1. BMC Genetics. 20. 10.1186/s12863-019-0748-6.

Long, X.Y., Balcerzak, M., Gulden, S., Cao, W., Fedak, G., Wei, Y.M., Zheng, Y.L., Somers, D., Ouellet, T. 2015. Expression profiling identifies differentially expressed genes associated with the Fusarium head blight resistance QTL 2DL in wheat. Physiology and Molecular Plant Pathology. 90: 1-11.

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