Genotyping-by-Sequencing for the Study of Genetic Diversity in Puccinia triticina .

Leaf rust, caused by Puccinia triticina Erikss., is globally the most widespread rust of wheat. Populations of P. triticina are highly diverse for virulence, with many different races found annually. The genetic diversity of P. triticina populations has been previously assessed using different types of DNA markers. Genotyping technologies that provide a higher density of markers distributed across the genome will be more powerful for analysis of genetic and phylogenetic relationships in P. triticina populations. In this study, we utilized restriction associated DNA (RAD) genotyping-by-sequencing (GBS) adapted for the Ion Torrent sequencing platform for the study of population diversity in P. triticina. A collection of 102 isolates, collected mainly from tetraploid and hexaploid wheat, was used. The virulence phenotypes of the isolates were determined on 20 lines of Thatcher wheat near isogenic for leaf rust resistance genes. Seven races were found among 57 isolates collected from tetraploid wheat, and 21 races were observed among 40 hexaploid wheat type isolates. This is the first study to report durum wheat virulent races to Lr3bg in Tunisia, Lr I 4a in Morocco, and Lr3bg and Lr28 in Mexico. Ethiopian isolates with high virulence to durum wheat but avirulent on Thatcher (hexaploid wheat) were tested for virulence on a set of durum (tetraploid) differentials. A subset of 30 isolates representing most of the virulence phenotypes in the 102 isolates were genotyped using RAD-GBS. Phylogenetic analysis of 30 isolates using 2,125 single nucleotide polymorphism (SNP) markers showed nine distinct clusters. There was a general correlation between virulence phenotypes and SNP genotypes. The high bootstrap values between clusters of isolates in the phylogenetic tree indicated that RAD-GBS can be used as a new genotyping tool that is fast, simple, high throughput, cost effective, and provides a sufficient number of markers for the study of genetic diversity in P. triticina.