Development of multiplex PCR to detect slow rust resistance genes Lr34 and Lr46 in wheat

Abstract

Leaf rust caused by Puccinia triticina belongs to one of the most dangerous fungal diseases of wheat (Triticum aestivum L.) and is the cause of large yield losses every year. Here we report a multiplex polymerase chain reaction (PCR) assay, which was developed for detection of two important wheat slow rust resistance genes Lr34 and Lr46, using two molecular markers: csLV34 and Xwmc44, respectively. The presence of genes was analyzed in one winter wheat variety TX89D6435 and five spring wheat varieties: Pavon F76, Parula ‘S’, Rayon 89, Kern, Mochis 88. Both Lr34 and Lr46 genes were identified in variety TX89D6435, gene Lr34 was also identified in Parula ‘S’ and Kern varieties, and gene L46 occurs in Pavon F76 and Mochis 88 variety. None of the resistance genes tested was detected in the Rayon 89 variety. The use of the multiplex PCR method allowed to shorten the analysis time, reduce costs of analyses, and reduce the workload.

Pathogen-induced alterations in methylome during Lr48-mediated APR in wheat (Triticum aestivum L.) 

“…In wheat, APR genes for leaf rust have been classi ed by some in two groups, one group including 7 genes (Lr12, Lr13, Lr22a/b, Lr35, Lr37, Lr48 and Lr49) that are race-speci c and hypersensitive and the other group having 8 genes (Lr34, Lr46, Lr67, Lr68, Lr74, Lr75, Lr77 and Lr78) that are race-nonspeci c and non-hypersensitive , 41,42 . Other workers recognized only the 8 non-race-speci c non-hypersensitive genes as APR genes 43,44 .…”

“…In order to understand the Lr48-mediated molecular events involved in downstream signal transduction and defense responses, transcriptome studies have also been conducted 8,9,13 . We believe that the expression of each APR gene as well as downstream signal transduction and defense responses might be partly regulated through epigenetic modi cations 44,45,46 . In particular, the role of DNA methylation in expression of APR genes for bacterial blight in rice was earlier examined, where a correlation was reported between DNA methylation (examined using MSAP) and repression of gene activity examined using Northern hybridization 44 .…”

Identification of leaf rust resistance genesLr34andLr46in common wheat (Triticum aestivumL. ssp.aestivum) lines of different origin using multiplex PCR

“…The PCR reaction was performed in a 20 µL volume of the mixture per sample, where 1 µL of the mixture constituted previously isolated DNA. Mixtures differed in the volume of individual components depending on the gene tested [ 16 ].…”

“…The 20 µL reaction mixture for gene Lr46 consisted of the following: 5× Green GoTaq® Flexi Buffer – 4 µL, MgCl 2 (2 mM) – 1.8 µL dNTP Mix (0.2 mM each) – 0.5 µL, GoTaq® DNA Polymerase (1.25 µ) – 0.25 µL, water – 11.05 µL, primers (0.25 µM) – 2 × 0.7 µL, and DNA template (50 ng/µ) – 1 µL. The PCR reaction profile for both genes differed in primer annealing temperature, determined on the basis of their melting temperature, and was as follows: initial denaturation – 5 min at 94°C, then 45 cycles (denaturation – 45 s at 94°C, annealing – 30 s at 55°C ( Lr34 ) or 59°C ( Lr46 ), synthesis – 1 min at 72°C), final synthesis – 7 min at 72°C, and storage at 4°C for a maximum of 24 h [ 16 ].…”

Development of Triticale × Wheat Prebreeding Germplasm With Loci for Slow-Rusting Resistance

“…The PCR reaction volume was 20 µl, consisting of 100 nM each of the two primers, 2 × TaqNova-DNA hot-start polymerase buffer (Blirt, Poland), and 50 ng of genomic DNA as template. A typical PCR procedure was as follows: 5 min at 95 • C, then 35 cycles of 30 s at 94 • C, 30 s at 50-60 • C (Skowrońska et al, 2019), 1 min at 72 • C, and 5 min at 72 • C. PCR products were run on 2% agarose gel (Lab Empire, Poland) with 1% Trisborate-ethylenediaminetetraacetic acid (TBE) buffer. DNA was visualized via Midori Green Direct (Nippon Genetics Europe, Germany) that was added to the samples.…”