The Lr34/Yr18/Sr57/Pm38/Ltn1 multi-resistance locus has been deployed and remained effective in wheat cultivars for more than 100 years. The durability and pleiotropic nature makes Lr34 a unique and highly valuable resource for rust resistance breeding. Despite its functional annotation as an ABC transporter, the mode of action is unknown. Considering this, we aimed to decipher molecular factors and signaling components essential for Lr34 function using RNA-seq of Chara resistant (Lr34) and Chara mutant (heavy ion irradiation, HII) susceptible wheat lines. Screening of Chara and Chara HII lines with Lr34-specific markers confirmed the integrity of Lr34 in both lines; however, phenotyping confirmed rust and powdery mildew susceptibility in the Chara HII lines. Plants were grown under controlled conditions and infected with Puccinia triticina pathotype 76-1,3,5,7,9,10,12,13+Lr37 at the flag leaf stage. Flag leaves were sampled at 0, 24, 48, 72, 96 and 168 hours post inoculation (hpi) from mock and infected plants. Based on real-time PCR analysis of basal defense genes and the Lr34 gene, we selected 72 hpi for RNA-seq with four biological replicates per condition. The samples were sequenced on an Illumina Hiseq 4000 at the Beijing Genomics Institute, China. A total of 9.0 Gb of sequence (2.25 Gb/library) from 16 libraries for four conditions was obtained. Differential expression analysis was performed using the Tuxedo analysis pipeline with standard parameters. Analysis revealed deletion of DNA fragments with collinear gene order on chromosomes 1A, 2D, 5A, 5B, 5D and 7D of Chara HII mutants. To determine the significance of the deletions we performed bulk segregant analyses on segregating F2 populations of Chara ? Chara HII crosses. Analyses revealed key genomic regions associated with Lr34-functional resistance and we are in the process of validating candidate genes using qPCR.
Deciphering the molecular factors essential for Lr34-mediated resistance in wheat
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