Validating Molecular Markers for Barley Leaf Rust Resistance Genes Rph20 and Rph24

Abstract

Improving resistance to barley leaf rust (caused by Puccinia hordei) is an important breeding objective in most barley growing regions worldwide. The development and subsequent utilization of high-throughput polymerase chain reaction (PCR) based co-dominant molecular markers remains an effective approach to select genotypes with multiple effective resistance genes, permitting efficient gene deployment and stewardship. The genes Rph20 and Rph24, which confer widely effective adult plant resistance (APR) to leaf rust, are common in European and Australian barley germplasm (often in combination), and act interactively to confer high levels of resistance. Here we report on the development and validation of co-dominant insertion-deletion (indel) based PCR markers that are highly predictive for the resistance alleles Rph20.ai and Rph24.an (both referred to as Rph20 and Rph24).

Characterization of Leaf Rust Resistance in International Barley Germplasm Using Genome-Wide Association Studies

“…Three APR genes, Rph20 [ 15 ], Rph23 [ 11 ] and Rph24 [ 16 ], are considered to be pathotype-non-specific and provide low-to-moderate levels of protection individually and moderate-to-high levels in combination [ 5 , 11 , 16 ]. Robust, closely linked markers are available for these three genes [ 19 ], and consequently they are being targeted more as sources of leaf rust resistance by barley breeders. Although these three APR genes have been shown to be race-non-specific thus far, it should not be presumed that this will always be the case, as APR can be race-specific [ 28 ].…”

“…To a quantity of 10 μL of PCR reaction mix was added 2 μL DNA, 2 μL 10 MiFi Buffer, 1μL of each forward and reverse primer (10 μM), 0.1 μL taq polymerase (Bioline), and 3.9 μL distilled autoclaved water. The PCR conditions used for the genotypic analysis using markers linked to Rph15 -, Rph20 -, Rph23 - and Rph24 -linked markers were previously described by Dracatos [ 23 ], Hickey et al [ 15 ], Singh et al [ 11 ] and Dracatos et al [ 19 ], respectively. Alleles of different sizes were resolved using 2% agarose gel that was prepared by dissolving 2 g agarose per 100 mL of 1× Tris-borate EDTA (TBE) buffer (90 mM Tris-borate + 2 mM EDTA-pH 8.0), and 1 μL of GelRed was added per 100 mL of gel solution for staining.…”

“…Molecular markers linked to rust resistance genes, where available, can further assist in the identification of resistance and the determination of whether they are different from those described previously. Very closely linked codominant markers are now available for all three APR genes [ 19 ], the ASR genes Rph7 [ 20 ] and Rph13 [ 21 ], and gene-based predictive markers have been developed for Rph3 [ 22 ] and Rph15 [ 23 ]. These markers are very useful for marker-assisted selection and validating new resistances.…”

Understanding the expression and interaction of Rph genes conferring seedling and adult plant resistance to Puccinia hordei in barley

“…The BC n F 3 families derived from crosses B+Rph11.p/Flagship and B +Rph14.ab/Flagship did not segregate (probably there was an accidental self-pollination instead of a cross) and were therefore incorrect and hence not studied further. The HR lines from each combination were selected for further studies because they represent putative homozygous gene combination stocks of each ASR gene in combination with the APR gene Rph20 (based on selection of BC 1 F 2 individuals using a codominant marker linked to Rph20 (Dracatos et al 2021); Fig. 1).…”

Registration of Golden Promise/Otis barley recombinant inbred line mapping population

“…In barley, RIL populations have been used for mapping genes associated with biotic stressors (Alhashel et al., 2021; Dracatos et al., 2019; Dracatos et al., 2021; Huang et al., 2018; Tamang et al., 2019) and abiotic stresses (Baum et al., 2003; Mikolajczak et al., 2017; Teulat et al., 1998; von Korff et al., 2008). We report a RIL population (MP‐3, NSL 545640 MAP) derived from two popular heritage barley cultivars, Golden Promise (GP) and Otis.…”