Characterization of Candidate Overwintering Genes in Forage: Biofuel Napiergrass (Pennisetum purpureum Schumach.) Charlie Dowling1, Byron Burson2, Jim Heitholt3, Dariusz Malinowski1, Esten Mason 4, and Russell Jessup1 1Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843-2474 2USDA-ARS, Crop Germplasm Research Unit, Texas A&M University, College Station, TX 77843-2474 3Department of Agriculture Sciences, Texas A&M University-Commerce, Commerce, TX 75429 4Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AK 72701 Overview Results and Discussion Perennial grasses are promising as renewable energy sources. Grasses with the highest rates of biomass accumulation are C4 tropical and subtropical species. Napiergrass (‘elephantgrass’; Pennisetum purpureum Schumach.) is a tropical, C4 grass with superior forage quality and yield. Perennial feedstocks like napiergrass allow for sustainable biofuel production via carbon sequestration, erosion control, and improved food security by utilization across marginal and abandoned lands. Similar to sugarcane, napiergrass is adapted predominately to the Southern United States (zone 8b; Figure 3). Traditional and genomic approaches are being utilized to select genotypes adapted to more temperate climates. Phenotypic selection criteria for improved overwintering included traits for tiller number, rhizome-derived shoots, rhizome distance from crown, and overall plant robustness. Screening of selections from a diverse germplasm collection in College Station, TX (zone 8b) were advanced to Commerce, TX during the 2012-2013 winter (zone 8a). The surviving genotypes were then screened the following winter in Vernon, TX (zone 7b). A comparative genomics strategy that identified annotated genes with putative overwintering-related function and high synteny across grass species was utilized. 2 out of 13 novel Taqman® hydrolysis probes showed up-regulation in absolute gene expression (Figure 4) Resulting up-regulation in Apetala 2-like (AP2-like) and Rare Cold Inducible 2-like (RCI2-like) transcripts AP2-like transcript on S. bicolor Chr. 2 (http://phytozome.org; E-value: 2.4 e-27) within 7 Mbp of rhizome QTL RCI2-like transcript on S. bicolor Chr. 9 (http://phytozome.org; E-value: 1.5 e-21) within 6 Mbp of rhizome QTL The AP2/Ethylene Response Factor (ERF) TF superfamily present in many plant tissues and is important for growth, development, and responses to biotic and abiotic stresses RCI2 is in 14-3-3 superfamily which are known to react to environmental stresses (cold, high salt, drought) RCI2-like genes shown to regulate freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling ethylene biosynthesis Phenotypic selections towards advancing napiergrass cold—tolerance adaptation feasible with sufficient plasticity in its genome TAMU Cold-Tolerant Napiergrass Adaptation (Vernon, TX; 2013-14) Proposed Northern Fringe of Adaptation for Cold Tolerant Napiergrass (Sollenberger, UF, 2012) TAMU Cold-Tolerant Napiergrass Adaptation (Commerce, TX; 2012-13) Figure 3. Texas A&M (TAMU) napiergrass adaptation over two selection cycles related to the proposed northern limit (blue line). The red star represents Commerce, TX (zone 8a) and the black star represents Vernon, TX (zone 7b). Initial selections made in College Station, TX (blue star). Cell Division Cell Elongation Genotype 1 Genotype 1 Figure 1. Napiergrass cold–tolerant genotypes that survived the 2013-2014 winter in Vernon, TX. Materials and Methods Cell Division Cell Elongation Fall 2013 (prior to winter dormancy) and Spring 2014 (after green-up) rhizome sampling (See Figure 2) Rhizome tissue in zones of cell elongation and division harvested separately and subjected to gene expression analysis via quantitative-PCR (qPCR) (Figures 2 & 4) Candidate genes selection criteria: 1) single or double allele dosage, 2) 100—200 bp product size, 3) targeted functional annotation (TFs upstream in cold response pathways), and 4) co-localization with S. bicolor rhizome and overwintering QTLs ROX™ Passive Reference Dye, ubiquitin, and β-tubulin for normalization of fluorescence and expression Genotype 2 Genotype 2 Figure 2. Plants subjected to non-destructive and destructive (shown) sampling of rhizomes in the Fall and Spring, respectively, Figure 4. Absolute transcript expression levels of two candidate genes for overwintering and cold—tolerance. Rhizomes of two napiergrass cold—tolerant genotypes sampled from the same plant in the Fall of 2013 and Spring of 2014 in Commerce, TX. Acknowledgements