Using RNA Sequencing and In Silico Subtraction to Identify Resistance Gene Analog Markers for Lr16 in Wheat

Leaf rust, caused by Puccinia triticina Eriks., is one of the most widespread diseases of wheat and breeding for resistance is one of the most effective methods of control. Lr16 is a wheat leaf rust resistance gene (R-gene) that provides resistance at both the seedling and adult stages. Simple-sequence repeat (SSR) markers have been used to map Lr16 to the distal end of chromosome 2B. The objectives of this study were to use RNA sequencing (RNA-seq) and in silico subtraction to identify new R-gene analogs (RGAs) and use them as Lr16 markers. RNA was isolated from the susceptible wheat cultivar Thatcher (Tc) and the resistant Tc isolines TcLr10, TcLr16, TcLr21, and sequenced using Illumina technology. Using in silico subtraction, sequences from the resistant Tc isolines were aligned to a Tc reference expressed sequence tag (EST) set. Sequences not aligning to the Tc reference were assembled into contigs and analyzed using BLASTx to determine putative gene functions. Primer pairs were designed for 181 RGA sequences, of which, 137 amplified in at least one of the parents. A mapping population was developed with 165 F₂ lines from a cross between the rust-susceptible cultivar Chinese Spring (CS) and TcLr16. Two RGA markers XTaLr16_RGA266585 and XTaLr16_RGA22128 were identified that mapped proximally 1.2 and 23.8 cM from Lr16, respectively. Three SSR markers Xwmc764, Xwmc661, and Xbarc35 mapped between these two RGA markers at distances of 5.0, 10.9, and 16.1 cM from Lr16, respectively. In silico subtraction is an effective technique for isolating RGAs linked to R-genes of interest.