Development and Characterization of Expressed Sequence Tag-Derived Microsatellite Markers for the Wheat Stem Rust Fungus <italic>Puccinia graminis</italic> f. sp. <italic>tritici</italic>

Zhong, S., Leng, Y., Friesen, T. L., Faris, J. D., and Szabo, L. J. 2009. Development and characterization of expressed sequence tag-derived microsatellite markers for the wheat stem rust fungus Puccinia graminis f. sp. tritici. Phytopathology 99:282-289. Puccinia graminis f. sp. tritici is the causal agent of stem rust disease in wheat. The rust fungus has caused devastating disease epidemics throughout history and is still posing a potential threat to wheat production in some regions of the world due to the appearance of new races. To develop microsatellite or simple sequence repeat (SSR) markers for use in population genetics studies, a total of 60,579 expressed sequence tag (EST) sequences (reads) generated from P. graminis f. sp. tritici were screened for tandemly repeated diand tri-nucleotide units using a bioinformatics approach and 708 unisequences containing putative SSR loci with six or more repeat units were identified. Flanking primers were designed for 384 unique SSR loci, which mapped to different locations of the draft genome sequence of the fungus. Of the 384 primer pairs tested, 72 EST-SSR markers were eventually developed, which showed polymorphism among 19 isolates of P. graminis f. sp. tritici and 4 isolates of P. graminis f. sp. secalis evaluated. Thirty-two of the SSR loci were also evaluated in three other rust fungi (P. triticina, P. hordei, and P. coronata f. sp. hordei) for cross-species transferability. These SSR markers derived from ESTs will be useful for characterization of population structures and for gene mapping in P. graminis. Wheat stem rust, caused by Puccinia graminis f. sp. tritici, has been one of the most devastating diseases in human history. In the early to mid 1950s, stem rust epidemics caused approximately 50% yield losses of wheat in North Dakota and Minnesota due to the emergence of a new race (race 15B) (21,22). Since 1974, stem rust has been under control in North America due to the incorporation and pyramiding of stem rust resistance genes into wheat cultivars. The barberry (Berberis vulgaris) eradication program, initiated in the early 1920s, also maintained the effectiveness of resistant genes longer by reducing the chance of the generation of new virulent races through sexual recombination of the fungus (22). However, wheat stem rust still poses a potential threat to wheat production worldwide. Especially, a new race (Ug99 or TTKS) and its variants were recently found in Eastern Africa and caused serious disease problems in that region (26,34). They have also triggered a serious concern to many other regions of the world where the stem rust resistance genes deployed in commercially grown wheat varieties were not effective against them (13–15). P. graminis f. sp. tritici is a heteroecious macrocyclic fungus with five spore stages (22). The uredia and telia are produced on wheat while the pycnia and aecia are formed on the alternate host, barberry (B. vulgaris). The uredinial stage is the most prevalent stage of the life cycle and can repeat on gramineous hosts under favorable conditions. In the late season, the rust pustules form telia, which produce teliospores. Meiosis occurs in teliospores and basidiospores emerge after teliospores germinate. Basidiospores can infect barberry plants and produce pycnia with pycniospores and receptive hyphae. The receptive hyphae from one mating type can be fertilized by a pycniospore of opposite mating type to produce aecia with dikaryotic aeciospores. The aeciospores infect cereals and reproduce urediniospores again. Significant variation within a P. graminis f. sp. tritici population can be generated through genetic recombination during this sexual cycle when barberry plants exist, although several other mechanisms also operate, such as mutation, somatic hybridization, and alien introduction (5). Virulence tests are commonly used to detect pathogen variation and a number of races have been identified. However, virulence markers are limited and subjected to host selection. Since the 1980s, molecular markers have been used to study the genetic variation and population structure of fungi, including P. graminis f. sp. tritici. Using enzyme markers combined with virulence evaluation, Burdon and Roelfs (6) compared the sexually and asexually reproducing populations of P. graminis f. sp. tritici and found that the sexually reproducing population exhibited higher genetic diversity compared with the asexually reproducing population. Other markers including randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were also used to analyze genetic diversity in wheat stem rust (24) and other rust fungi (8,20). Although these markers provide powerful tools for population genetic studies, each of them has limitations in application. For the dikaryotic organisms like Puccinia species, codominant markers such as SSRs are more informative in revealing genetic variations when compared to dominant markers (30). SSR markers have been developed for many species of plants and fungi and are usually developed from genomic DNA through the construction of SSR-enriched libraries. This approach, although effective, is quite time consuming and labor-intensive. In recent years, more and more expressed sequence tags (ESTs) from cDNA libraries have been generated for a number of organisms, Corresponding author: S. Zhong; E-mail address: [email protected] doi:10.1094 / PHYTO-99-3-0282 © 2009 The American Phytopathological Society