De novo assembly of giant reed (Arundo donax L.) leaf transcriptome using RNA-Seq provides insight into drought response, gene discovery and genetic marker identification
Author(s)
Evangelistella, Chiara
Date Issued
June 6, 2017
Type
Doctoral Thesis
Abstract
Introduction
Arundo donax has attracted renewed interest as a potential candidate bioenergy crop for use in biomass-to-liquid fuel-conversion processes and biorefineries. This is due to its high productivity, adaptability to marginal lands, among which drought-prone environments, and suitability for biofuel and biomaterial production. Despite its importance, currently the genomic resources publicly available for supporting the improvement of this species are still limited and also there is little information about its molecular response to drought in field.
Results
Illumina next-generation mRNA-sequencing (mRNA-Seq) was used to de novo assemble and characterize the A. donax leaf transcriptome. The sequencing generated 1,249 million clean reads that were assembled using single-k-mer (SK) and multi-k-mer (MK) approaches into 62,596 unique sequences (unitranscripts) with an N50 of 1,134 bp. TransDecoder and Trinotate software suites were used to obtain putative coding sequences and to annotate them by mapping to UniProtKB/Swiss-Prot and UniRef90 databases, searching for known transcripts, proteins, protein domains and signal peptides. Furthermore, the unitranscripts were annotated by mapping them to the NCBI non-redundant, GO and KEGG pathway databases using Blast2GO software. The transcriptome was also characterized by customized BLAST searches to investigate homologous transcripts of key genes involved in important metabolic pathways, such as lignin, cellulose, purine and thiamine biosynthesis and carbon fixation. Moreover, a set of homologous transcripts of key genes involved in stomatal development and of genes coding for stress-associated proteins (SAPs) was identified. Additionally, 8,364 simple sequence repeat (SSR) markers were identified that represent the first genetic marker catalog of A. donax. Furthermore, 53 SSRs (PolySSRs) were then predicted to be polymorphic between ecotype-specific assemblies, suggesting a certain degree of genetic variability in the studied A. donax ecotypes (called EcoA, EcoB and EcoC). Furthermore, differentially expressed transcripts (DETs) between well-watered (WW) and natural moderate drought stress (mDr) conditions were identified in three A. donax ecotypes and three different timepoints (referred as T1, T2 and T3). All the three ecotypes over-expressed transcripts encoding for PFP and JIPs proteins, and employed the ABA (abscisic acid)-dependent pathways to cope with drought stress, several differentially regulated transcripts belonging to different transcription factors (TFs) families (e.g. NAC, WRKY, MYC, MYB, AP2/ERF, bZIP, FAR, FRS, bHLH, PIF) and encoding for proteins involved in ABA perception (e.g. PP2C, PYL4-like and PYR1-like). The differential regulation of FAR1 and FRS genes in EcoA and EcoB could be associated with a greater control of stomata size, thereby of water loss, and thus suggesting a greater ability to tolerate
drought stress compared to EcoC. In addition, in EcoA the down-regulation of SDD1 gene, together with the up-regulation of FAR1 transcript, could be associated with a molecular adaptive response of this ecotype which controls stomatal density and size to fine-regulate water flow and gas exchange under drought stress. Furthermore, all the three ecotypes down-regulated different AQP genes (e.g. NIP2;2 and NIP 1;1-like; TIP1;1 and TIP4;2; SIP2;1 and PIP1;1 and PIP2;7) in order to regulate water flow across cells and subcellular compartments. Finally, for EcoC transcripts encoding for a TNKS1-like proteins were up-regulated, suggesting a role of this gene, which is involved in DNA damage repair, cell death pathways, transcription regulation and chromatin modification/remodeling, in the drought stress response of this ecotype. In addition, for EcoA and EcoB the down-regulation of genes involved in the lignin biosynthetic pathway was observed, which represents an important result in the context of A. donax as bioenergy crop for biofuel production. Finally, in EcoA and EcoB the down-regulation of EIL3 gene could be associated with a delay in the progression of drought-induced leaf senescence and, a possible increase in water stress tolerance in these ecotypes.
Conclusions
This study provides the first publicly available transcriptome resource for A. donax bioenergy crop. The functional annotation and characterization of the leaf transcriptome will be highly useful for providing insight into the molecular mechanisms underlying the extreme adaptability of A. donax. The identification of homologous transcripts involved in key metabolic pathways offers a platform that will direct future efforts in genetic improvement of this species. In addition, the identified SSRs will facilitate the harnessing of untapped genetic diversity. This transcriptome should be of value to ongoing functional genomics and genetic studies in this crop of paramount economic importance. Finally, differential expression (DE) analysis allowed the identification of potential candidate genes useful for accelerating future molecular breeding program aiming to obtain increasingly drought tolerant A. donax ecotypes.
Additional information
Dottorato di ricerca in Scienze, tecnologie e biotecnologie per la sostenibilità
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