| dc.description.abstract | Switchgrass (Panicum virgatum L.) is a perennial warm season grass native to a large portion of North America. Because of its low input requirements, broad adaptation, and high yield potential, switchgrass has been used for forage production and soil conservation and is considered an ideal renewable biomass feedstock crop for biofuel production. However, switchgrass can be affected by several fungal diseases. One of these is rust, caused by Puccinia emaculata, an obligate parasitic fungus. Puccinia emaculata has been observed on agronomic switchgrass causing chlorosis and necrosis of leaf tissues, lodging, and plant death, reducing feedstock quality and biomass yield of switchgrass up to 60% and 50%, respectively. Currently, little is known about switchgrass rust, and its unclear etiology complicates the development of effective management strategies. In order to better understand the biology of the pathogen, this study focused on two main objectives: 1) generate a multilocus phylogeny to determine the species' phylogenetic status and to assess its genetic diversity across five US states (MS, OK, VA, IA, and SD) and 2) develop and characterize de novo simple sequence repeats (SSRs) and expressed sequence tag (EST)-SSRs to study the population biology of P. emaculata. Since DNA from bulked urediniospores produced mixed templates, PCR products of three phylogenetically informative genomic loci, the ribosomal internal transcribed spacer (ITS) region, and the β-tubulin (bTub) and translation elongation factor-1α (TEF1a) genes, were cloned prior to sequencing. Differences in haplotype diversity was observed among loci (ITS=13; bTub=24; TEF1a= 27), with large variation in the patterns of distribution of haplotypes across states. Distribution of bTub and TEF1a haplotypes were mostly local, while ITS haplotypes were distributed both across multiple states and locally. In order to perform multilocus phylogenetic analyses, a single spore whole genome amplification (ssWGA) protocol was standardized, which produced sufficient single cell DNA for PCR of single copy genes. Single gene and multilocus phylogenies supported the monophyletic status of P. emaculata. Using a P. emaculata di- and trinucleotide repeat-enriched library, 49 SSR loci were identified, of which 8 were informative for multispore DNA samples and 6 were informative for a collection of 25 ssWGAs from five states. To develop EST-SSRs, RNA was isolated from germinated and non-germinated urediniospores from OK and VA using a novel modified method, cDNAs were generated and submitted for RNA sequencing (RNA-Seq). Thirty three EST-SSRs were identified, 12 of which were informative for a multistate collection of 25 ssWGAs. Genetic diversity was observed across single spore collections from five states using 18 microsatellite loci. Future studies will examine genetic variation, population structure and pathogenicity variation among multistate P. emaculata populations. | |
