Investigation of Un8-Mediated Barley Loose Smut Resistance
Date
2017-07-04
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Degree Level
Doctoral
Abstract
Barley loose smut, caused by the basidiomycete pathogen Ustilago nuda (Jens.) Rostr. (U. nuda), is a common fungal disease throughout Canadian barley growing regions and can be effectively controlled by the Un8 resistance gene. The first study (Chapter 3) was designed to isolate the Un8 gene by map-based cloning. The Un8 gene was delimited to a 0.108 cM interval on chromosome arm 1HL and a minimal tiling path consisting of two overlapping bacterial artificial chromosomes was identified. Sequence analysis identified a Un8 candidate gene predicted to be a putative protein kinase with two kinase domains. Twenty-six cultivated and eight wild barley accessions with diverse genetic backgrounds were collected for the second study (Chapter 4) and sequence alignment revealed that all resistant accessions from Canada shared the same amino acid sequence with the landrace accession, CN91953, which was reported as the donor of Un8 to North American barley breeding programs.
The remaining three studies focused on elucidating the mechanisms underlying Un8-mediated loose smut resistance. In Chapter 5, a simple and reliable diagnostic method was developed to examine the infection processes of U. nuda within barley seeds and it was found that the early seedling stage might be the most important time point for Un8-conditioned loose smut resistance. Seedling mortality was also observed in resistant and susceptible lines which led to questions as to whether this was a function of the high inoculum concentration used to evaluate disease response. To further investigate this resistance, the commonly used inoculation method was improved by reducing the inoculum concentration (Chapter 6). During this study a large fitness cost (i.e. seedling mortality) previously observed was associated only with resistant lines carrying the Un8 gene. In the final study (Chapter 7), expression analysis was undertaken to better understand Un8-mediated resistance and seedling mortality observed. Two barley genes involved in cytokinin regulation, CKX1 and CKX2.1, which encode cytokinin oxidase/dehydrogenase (CKX) enzymes to irreversibly degrade cytokinins, were significantly up-regulated at time points that coincided with early seed germination. This indicated that the cytokinin pathway may be involved in the loose smut resistance conditioned by the Un8 gene.
Taken together, this study has provided deeper insight into the long-lived Un8 loose smut resistance gene, including a possible role for cytokinins in barley loose smut resistance.
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Keywords
barley, loose smut, Un8, map-based cloning, protein kinase, seedling mortality, inoculum concentration, cytokinin
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Degree
Doctor of Philosophy (Ph.D.)
Department
Plant Sciences
Program
Plant Science