An Improved Detached-Leaf Assay for Phenotyping Net Blotch of Barley Caused by Pyrenophora teres.

Net blotch, caused by Pyrenophora teres, is a major barley (Hordeum vulgare) leaf disease worldwide. P. teres occurs as two forms-P. teres f. teres, and P. teres f. maculata-inducing net and spot-like symptoms, respectively. An intact-seedling assay, where entire seedlings are inoculated by spraying with a conidial suspension, is frequently used for phenotyping net blotch. However, this presents a biosecurity risk in the glasshouse when nonlocal isolates are being screened. Alternatively, a detached-leaf assay (DLA-droplet method) can be used in which leaf segments laid out in a covered tray are inoculated with droplets of a conidial suspension, confining the inoculum. However, using this method, net and spot form symptoms cannot be distinguished from each other. We have developed an improved DLA (DLA-spray method) in which detached whole leaves are sprayed with the inoculum to produce distinct lesions. We compare the results for the three phenotyping methods above using four isolates from both net and spot forms of the disease to inoculate a standard set of eight barley genotypes. Results indicate that the DLA-spray method is a functional, informative and rapid test that readily differentiates the two forms of the pathogen in a biosecure environment.

Investigating successive Australian barley breeding populations for stable resistance to leaf rust.

KEY MESSAGE: Genome-wide association studies of barley breeding populations identified candidate minor genes for pairing with the adult plant resistance gene Rph20 to provide stable leaf rust resistance across environments. Stable resistance to barley leaf rust (BLR, caused by Puccinia hordei) was evaluated across environments in barley breeding populations (BPs). To identify genomic regions that can be combined with Rph20 to improve adult plant resistance (APR), two BPs genotyped with the Diversity Arrays Technology genotyping-by-sequencing platform (DArT-seq) were examined for reaction to BLR at both seedling and adult growth stages in Australian environments. An integrated consensus map comprising both first- and second-generation DArT platforms was used to integrate QTL information across two additional BPs, providing a total of four interrelated BPs and 15 phenotypic data sets. This enabled identification of key loci underpinning BLR resistance. The APR gene Rph20 was the only active resistance region consistently detected across BPs. Of the QTL identified, RphQ27 on chromosome 6HL was considered the best candidate for pairing with Rph20. RphQ27 did not align or share proximity with known genes and was detected in three of the four BPs. The combination of RphQ27 and Rph20 was of low frequency in the breeding material; however, strong resistance responses were observed for the lines carrying this pairing. This suggests that the candidate minor gene RphQ27 can interact additively with Rph20 to provide stable resistance to BLR across diverse environments.

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm.

KEY MESSAGE: "To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants." Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.

Mapping Rph20: a gene conferring adult plant resistance to Puccinia hordei in barley.

A doubled haploid (DH) barley (Hordeum vulgare L.) population of 334 lines (ND24260 × Flagship) genotyped with DArT markers was used to map genes for adult plant resistance (APR) to leaf rust (Puccinia hordei Otth) under field conditions in Australia and Uruguay. The Australian barley cultivar Flagship carries an APR gene (qRphFlag) derived from the cultivar Vada. Association analysis and composite interval mapping identified two genes conferring APR in this DH population. qRphFlag was mapped to the short arm of chromosome 5H (5HS), accounting for 64-85% of the phenotypic variation across four field environments and 56% under controlled environmental conditions (CEC). A second quantitative trait locus (QTL) from ND24260 (qRphND) with smaller effect was mapped to chromosome 6HL. In the absence of qRphFlag, qRphND conferred only a low level of resistance. DH lines displaying the highest level of APR carried both genes. Sequence information for the critical DArT marker bPb-0837 (positioned at 21.2 cM on chromosome 5HS) was used to develop bPb-0837-PCR, a simple PCR-based marker for qRphFlag. The 245 bp fragment for bPb-0837-PCR was detected in a range of barley cultivars known to possess APR, which was consistent with previous tests of allelism, demonstrating that the qRphFlag resistant allele is common in leaf rust resistant cultivars derived from Vada and Emir. qRphFlag has been designated Rph20, the first gene conferring APR to P. hordei to be characterised in barley. The PCR marker will likely be effective in marker-assisted selection for Rph20.