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Title: Using RNA-sequencing and in silico subtraction to identify resistance gene analog markers for Lr16 in wheat

item REZAC-HARRISON, NICOLE - Kansas State University
item FRITZ, ALLAN - Kansas State University
item GLASSCOCK, JARRET - Cofactor Genomics
item AHMED, SARA - Cofactor Genomics
item MESSINA, DAVID - Cofactor Genomics
item Fellers, John

Submitted to: The Plant Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/4/2015
Publication Date: 7/10/2015
Publication URL:
Citation: Rezac-Harrison, N., Fritz, A.K., Glasscock, J., Ahmed, S., Messina, D.N., Fellers, J.P. 2015. Using RNA-seq and in silico subtraction to identify resistance gene analog markers for Lr16 in wheat. The Plant Genome. 8(2):1-9. doi: 10.3835/plantgenome2014.08.0040.

Interpretive Summary: Leaf rust is a common wheat disease that is found all over the world. In the United States, the most cost-effective way to combat the disease is to use resistance genes. One of these resistance genes, Lr16, provides resistance to many races of leaf rust and is desirable in wheat breeding programs. Since it is difficult to accurately score resistance using visual methods, plant breeders need DNA markers that they can use to follow Lr16 in their breeding programs. This paper describes a new technique to develop markers that takes advantage of the similarities among resistance genes and new high throughput DNA sequencing technology. A new DNA marker was developed for Lr16 that is easy to use and expected to be efficient for identifying resistant plants.

Technical Abstract: Leaf rust, caused by Puccinia triticina Eriks., is one of the most widespread diseases of wheat worldwide and breeding for resistance is one of the most effective methods of control. Lr16 is a wheat leaf rust resistance gene that provides resistance at both the seedling and adult stages. Simple sequence repeat (SSR) markers have been previously used to map Lr16 to the distal end of chromosome 2B. The objectives of this study were to use RNA-seq and in silico subtraction, identify new resistance gene analogs (RGA), and use them as markers for Lr16. RNA was isolated from the susceptible wheat cultivar ‘Thatcher’ (Tc) and the resistant Thatcher isolines TcLr10, TcLr16, TcLr21 and sequenced using Illumina technology. Using in silico subtraction, sequences from the Tc isolines were aligned to a Thatcher reference EST set. Sequences that did not align to the Tc reference were assembled into contigs and analyzed using BLASTx to determine putative gene functions. Primer pairs for 181 RGA sequences were designed, of which, 137 amplified in at least one of the parents. A population was developed for mapping of 201 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 1.2 cM 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 cM, 10.9 cM, and 16.1 cM from Lr16, respectively. In silico subtraction is an effective technique for isolating RGAs linked to resistance genes of interest.