Study : De Novo Genome Assembly of Guinea Grass Exposed to Elevated CO2 and Temperature


De Novo Genome Assembly of Guinea Grass Exposed to Elevated CO2 and Temperature
Introduction : By mid-century, global atmospheric carbon dioxide concentration ([CO2]) is predicted to reach 600 u­mol mol-1 with global temperatures rising by 2ºC. Rising [CO2] and temperature will alter the growth and productivity of major food and forage crops across the globe. Although the impact is expected to be greatest in tropical regions, the impact of climate-change has been poorly studied in those regions. Objectives : This experiment aimed to understand the effects of elevated [CO2] (600 umol mol-1) and warming (+ 2°C), singly and in combination, on Panicum maximum Jacq. (Guinea grass) metabolite and transcript profiles. Methods: We created a de novo assembly of the Panicum maximum transcriptome. Leaf samples were taken at two time points in the Guinea grass growing season to analyze transcriptional and metabolite profiles in plants grown at ambient and elevated [CO2] and temperature, and statistical analyses were used to integrate the data. Results: The MiSeq library was quantified by qPCR and sequenced on one MiSeq flowcell for 301 cycles using paired-end sequencing. HiSeq paired-end sequencing was done with four quantified libraries per treatment which were pooled in equimolar concentration, and sequenced on two lanes for 161 cycles. The final read lengths for MiSeq and HiSeq were 300 nt and 160 nt in length. A total of 635,649,277 reads were assembled from the MiSeq/HiSeq pools. Quality control for reads generated from sequencing was performed using FastQC. Quality reads were used to perform de novo transcriptome assembly using Trinity. The initial assembly consisted of 187,216 genes. A filter was applied to keep only those genes that had at least 10 reads (across the 4 replicates) for an individual treatment. The resulting transcriptome contained 45,073 genes and reads. Functional annotation of the genes was done by using BLAST against Arabidopsis thaliana, Zea mays, and Setaria italica. Overall design: 32 sample (n=4). Panicum maximum Jacq. (Guinea Grass) was planted in a complete replicated design at University of São Paul in Ribeirão Preto, São Paulo Brazil. 4 ambient temperature and [CO2] (C) plots, 4 elevated CO2 [600 ppm] and ambient temperature (eC) plots, 4 ambient [CO2] and elevated temperature (eT) [+ 2°C] plots, and 4 combined treatment (elevated [CO2] and elevated temperature (eCeT)) plots. Each plot was 2-m in diameter; [CO2] was controlled using Free Air CO2 Enrichment and canopy warming was provided by infrared ceramic heaters as described by Britto de Assis Prado et al. (2016). Treatment was started on April 22, 2014 in a 2500 m2 field. Samples for untargeted metabolomics (with GC-MS), de novo transcriptomics, and RNASeq, were taken 30 and 50 days post treatment exposure (May 22, 2014 and July 14, 2014). Lyophilized leaf material was sent to the University of Illinois Urbana-Champagin (Ainsworth lab) for the untargeted metabolomics, transcript assembly and RNASeq.


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