Unraveling the Genetics of Two Key Biomass Traits that Differentiate Upland and Lowland Tetraploid Switchgrass Ecotypes, Colonization by Mycorrhizal Fungi and Frost Tolerance. Project Summary The primary objectives of the project are to determine if symbiosis by arbuscular mycorrhizal fungi (AMF) alleviates the effects of cold-temperature stress in switchgrass, to identify quantitative trait loci (QTL) for susceptibility of switchgrass roots to colonization by AMF and for frost tolerance, and to identify candidate genes underlying these QTL. Switchgrass, a perennial grass native to the US, can be largely classified into two ecotypes, lowland and upland. The two ecotypes vary by a number of morphological, developmental and physiological characteristics including plant architecture, biomass production, spring regrowth, flowering time and frost tolerance. While lowland switchgrass produces higher biomass yields in its region of adaptation, the Southern US, this yield advantage is lost when lowland ecotypes are grown at more northern latitudes mainly because of high levels of winter kill. Understanding the genetic basis of frost tolerance is imperative to develop switchgrass varieties that combine the high yield potential of lowland ecotypes with the frost tolerance of upland ecotypes. Symbiosis with AMF enhances biomass production of switchgrass, in particular when grown under abiotic stress conditions such as drought and low soil fertility. AMF can also alleviate the effects of cold-temperature stress in many plant species, but such an effect has not yet been investigated in switchgrass. Preliminary analysis of AMF colonization levels in F1 progeny derived from a cross between the two ecotypes has identified chromosome regions from both parents that, when combined, give high susceptibility to AMF colonization. We will test whether plants with high levels of AMF colonization have higher survival rates under different freezing regimes relative to plants that have negligent colonization levels and relative to clonal plants grown in the absence of AMF. We will combine traditional QTL analyses with an expression QTL (eQTL) analysis focusing specifically on genes that are differentially expressed between an upland and lowland accession under cold-acclimatization and AMF inoculation to dissect susceptibility to AMF colonization and frost-tolerance into their genetic components and to identify candidate genes for those traits. Because we aim to map traits that vary significantly between ecotypes but are likely to be fixed within ecotypes, we will generate a new mapping population by crossing two F1 sibs that are heterozygous at all loci for lowland and upland alleles and that differ significantly in their susceptibility to AMF colonization. This population, which will be available at the start of the project, will be mapped using genotyping-by-sequencing (GBS) and form the framework for the QTL analyses. Clonal replicates of the parents and progeny, grown in the presence of AMF in both the glasshouse and in the field, will be subjected to freezing tests after cold-acclimatization. AMF colonization levels will be determined by conducting a metagenomic analysis of the microbial populations present within the roots of the mapping progeny. The transcriptomes of the mapping progeny (3 biological replicates) will be analyzed by sequencing (RNA-Seq) a minimum of 10 million reads per progeny. Phenotypic and expression data will be used in QTL analyses to identify regions of the genome and putative genes that contribute to AMF susceptibility and frost-tolerance. Sequence analysis of P. rudgei, an E genome species closely related to the A genome of switchgrass, will allow us to determine the subgenome origin of genes of interest and hence provide information on the relative contribution of the two switchgrass subgenomes (A and B) to traits. The project will generate the knowledge and biological materials needed to enhance the cold-tolerance of lowland switchgrass and will provide a set of resources that will greatly benefit the Switchgrass and Biofuels Communities. A 5,000 locus genetic map will assist with improving the switchgrass genome assembly. The extensive transcriptome data, featuring many ecotype-specific expression patterns, will provide an extremely valuable resource to extend the studies described in this proposal to other ecotype-specific traits. Project Participants Katrien M. Devos (PI) - Institute of Plant Breeding, Genetics and Genomics, and Dept. of Plant Biology, University of Georgia, Athens, GA, USA Jeffrey L. Bennetzen (Co-PI) - Dept. of Genetics, University of Georgia, Athens, GA, USA Paul Schliekelman (Co-PI) - Dept. of Statistics, University of Georgia, Athens, GA, USA Ali Missaoui (Co-PI) - Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA, USA Orville Lindstrom (Co-PI) - Dept. of Horticulture, University of Georgia, Griffin, GA, USA
