Analysis of the Barley Cereal Cyst Nematode Resistance Locus Rha2

Abstract

Cereal cyst nematodes (CCN, Heterodera avenae) are obligate soil-borne pathogens of cereal crops such as barley (Hordeum vulgare) that can reduce plant vigour and crop yield. Juvenile nematodes infect roots, establish feeding sites in root vascular tissue and become sedentary. Female nematodes mature into egg-filled cysts, which remain in the soil. Production of susceptible cultivars allows the numbers of cysts to increase, increasing the risk of damage to future crops. Some barley cultivars are resistant. Their roots can be infected, but few nematodes mature and form cysts. The focus of this thesis is the resistance conferred by the Rha2 locus on chromosome 2H of barley. By undertaking genotyping-by-sequencing of the resistant cultivars Sloop SA and Sloop VIC, their susceptible progenitor Sloop and other material, new DNA sequence polymorphisms were discovered on chromosome 2H. Marker assays were designed for these polymorphisms and assayed on two populations (Clipper/Sahara 3771 and Chebec/Harrrington) in which Rha2 had been previously mapped. An initial candidate region of 5,077 kbp was defined on the barley genome assembly. In order to obtain new recombinants and develop near-isogenic lines, Sloop SA was backcrossed to Sloop. Over 9,000 BC2F2 seeds were genotyped. A detailed genetic map of the candidate region was made by genotyping and phenotyping 64 selected BC2F3 families. The candidate region was narrowed to 978 kbp. That region of the genome assembly has 19 predicted genes. Markers in the region were evaluated on a range of barley germplasm and two markers were found to be diagnostic of CCN resistance. An RNA-seq experiment was conducted with root tissue sampled over a period of 28 days. The samples comprise non-inoculated control plants of the susceptible cultivar Sloop and inoculated plants of Sloop and its resistant derivatives Sloop SA and Sloop VIC. Of the 19 predicted genes in the candidate region, one gene (HORVU2Hr1G097780), which is annotated as encoding a tonoplast intrinsic protein, exhibited differential expression between the inoculated resistant cultivars and the susceptible cultivar. Further research is required for the functional characterisation of this gene. Interaction between cereal cyst nematodes and barley roots was also investigated by using laser ablation tomography to scan infected segments. On average, feeding sites in the roots of susceptible plants were smaller than those in the roots of resistant plants. The feeding sites in the roots of susceptible plants were surrounded by multiple dense layers of small cells. In contrast, the feeding sites in the roots of resistant plants were surrounded by layers of larger cells. This work presents a detailed genetic map of the Rha2 region of chromosome 2H, including two markers that appear to be diagnostic of resistance, the results of a transcriptomic experiment to explore differentially regulated genes, including candidate resistance genes. Laser ablation tomography was conducted on infected root tissue. Feeding site structure differed between a susceptible cultivar and its resistant derivative, including a smaller volume for the latter. The outcomes of this thesis research may lead to identification of the causal Rha2 resistance gene against cereal cyst nematode pathotype Ha13.