Date of Award

Fall 2018

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Dr. Iago Hale

Second Advisor

Dr. Thomas M. Davis

Third Advisor

Dr. Matthew MacManes

Abstract

Stem rust, caused by the macrocyclic fungal pathogen P. graminis (Pg), is one of the most devastating diseases of wheat and other small grains globally; and the emergence of new stem rust races virulent on deployed resistance genes brings urgency to the discovery of more durable sources of genetic resistance. Given its intrinsic durability and effectiveness across a broad range of pathogens, non-host resistance (NHR) presents a compelling strategy for achieving long-term rust control in wheat. However, NHR to Pg (Pg-NHR) remains largely unexplored as a protection strategy in wheat, in part due to the challenge of developing a genetically tractable system in which Pg-NHR segregates. In this dissertation, an investigation of Pg-NHR is undertaken via the pathogen's alternate (sexual) host, barberry (Berberis spp.). Within the highly diverse Berberis genus, numerous species function as alternate hosts to Pg but others are non-hosts. European barberry (B. vulgaris L.), for example, is susceptible to Pg infection but Japanese barberry (B. thunbergii DC.) is a non-host. In this study, the nothospecies B. ×ottawensis C.K. Scheid, an inter-specific hybrid between Pg-susceptible B. vulgaris and Pg-resistant B. thunbergii, is explored as a possible means of mapping the gene(s) underlying the apparent Pg-NHR exhibited by B. thunbergii. The overall goal of this research is to contribute to the global search for novel sources of potentially durable stem rust resistance genes.

The first chapter describes a field study conducted in western Massachusetts, in which a natural population of B. ×ottawensis was characterized to determine if the hybrid can be used to genetically dissect the Pg-NHR exhibited by B. thunbergii. A population of 63 B. ×ottawensis individuals were clonally propagated, phenotyped for disease response to Pg via controlled inoculation using overwintered telia of Pg found on naturally infected E. repens, and genotyped using the de novo genotyping-by-sequencing (GBS) pipeline GBS-SNP-CROP. Controlled inoculation of a subset of 53 B. ×ottawensis accessions, verified via GBS to be true, first-generation hybrids, revealed 51% susceptible, 33% resistant, and 16% intermediate phenotypes. Although such variation in disease response within a natural population of F1 hybrids could be explained by non-nuclear (cytoplasmic) inheritance of resistance, a similar pattern of segregation was observed in a population of B. ×ottawensis full-sibs, developed via controlled crosses. The results of this first chapter demonstrate not only that the Pg-NHR observed in B. thunbergii segregates among F1 interspecific hybrids with Pg-susceptible B. vulgaris but that the resistance is likely nuclearly inherited. Therefore, at least in principle, the gene(s) underlying Pg-NHR in B. thunbergii should be mappable in an F1 population derived from the controlled hybridization of the two parental species.

Building on the results of first chapter, the second chapter of this dissertation details the generation and use of a bi-parental B. ×ottawensis mapping population to develop genetic linkage maps for both parental species and begin mapping the gene(s) underlying Pg-NHR in B. thunbergii. Using 162 full-sib F1 hybrids and a total of 15,411 sequence variants (SNPs and indels) identified between the parents via GBS, genetic linkage maps with 1,757 and 706 markers were constructed for B. thunbergii accession 'BtUCONN1' and B. vulgaris accession 'Wagon Hill', respectively. In each map, the markers segregated into 14 linkage groups, in agreement with the 14 chromosomes present in these Berberis spp. The total lengths of the linkage maps were 1474 cM (B. thunbergii) and 1714 cM (B. vulgaris), with average distances between markers of 2.6 cM and 5.5 cM. QTL analysis for Pg resistance led to the identification of a single QTL, dubbed QPgr-3S, on the short arm of chromosome 3 of B. thunbergii. The peak LOD score of QPgr-3S is 28.2, and the QTL spans 13 cM, bounded by the distal SNP marker M411 and proximal SNP marker M969. To gain further insight into the QPgr-3S region, a chromosome-level 1.2 Gb draft genome for B. thunbergii was assembled using long PacBio reads and Hi-C data. By anchoring the B. thunbergii linkage map to the draft genome, the 13 cM QPgr-3S region was found to correspond to ~3.4 Mbp, represented by 10 contigs. Using a 189.3 Mb transcriptome assembled from a multiple tissue library of RNA-seq data, the QPgr-3S region was found to contain 99 genes. To help narrow this list to candidate genes of highest priority for subsequent investigation, a combination of approaches was taken. Specifically, annotation of the QTL region and differential gene expression analysis led to the identification of 12 candidate genes within the region. Of those, two emerge as particularly noteworthy due to the fact that both appear to be differentially expressed in B. thunbergii during Pg inoculation and belong to families implicated in disease resistance in other plant-pathogen systems, namely leucine-rich repeat receptor-like kinases (gene GG9708) and zinc ion binding SSM4 proteins (gene GG9868).

The final chapter provides a summary of the research and offers recommendations for future studies, building on the results to date. The original vision of this research was to explore the possible use of a heretofore uncharacterized system in identifying novel sources of resistance to Pg, and much progress has been made toward this end. Significant follow up research is needed, however, and the objectives of that research should be to validate, further characterize, and continue to dissect the QPgr-3S region; test candidate gene hypotheses; and seriously confront the difficult question of the relevance of gene(s) regulating Pg-NHR in B. thunbergii to durable strategies of stem rust resistance in wheat.

This dissertation demonstrates the feasibility of dissecting the genetics of a source of Pg-NHR in a novel way, using the interspecific hybrid barberry B. ×ottawensis. The genetic linkage maps developed in this work are the first available for any species within the ancient plant family Berberidaceae; and the draft genome is the first available for any plant within the order Ranunculales. Altogether, the resources developed in this study not only establish B. ×ottawensis as a viable system to dissect Pg-NHR but also makes available valuable resources for global rust surveillance work and ornamental horticulture breeding.

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