Development of an integrated transcript sequence database and a gene expression atlas for gene discovery and analysis in switchgrass (Panicum virgatum L.)

Abstract

SUMMARYSwitchgrass (Panicum virgatum L.) is a perennial C4 grass with the potential to become a major bioenergy crop. To help realize this potential, a set of RNA-based resources were developed. Expressed sequence tags (ESTs) were generated from two tetraploid switchgrass genotypes, Alamo AP13 and Summer VS16. Over 11.5 million high-quality ESTs were generated with 454 sequencing technology, and an additional 169 079 Sanger sequences were obtained from the 5′ and 3′ ends of 93 312 clones from normalized, full-length-enriched cDNA libraries. AP13 and VS16 ESTs were assembled into 77 854 and 30 524 unique transcripts (unitranscripts), respectively, using the NEWBLER and PAVE programs. Published Sanger-ESTs (544 225) from Alamo, Kanlow, and 15 other cultivars were integrated with the AP13 and VS16 assemblies to create a universal switchgrass gene index (PviUT1.2) with 128 058 unitranscripts, which were annotated for function. An Affymetrix cDNA microarray chip (Pvi_cDNAa520831) containing 122 973 probe sets was designed from PviUT1.2 sequences, and used to develop a Gene Expression Atlas for switchgrass (PviGEA). The PviGEA contains quantitative transcript data for all major organ systems of switchgrass throughout development. We developed a web server that enables flexible, multifaceted analyses of PviGEA transcript data. The PviGEA was used to identify representatives of all known genes in the phenylpropanoid-monolignol biosynthesis pathway.

A Genomics Approach to Deciphering Lignin Biosynthesis in Switchgrass

“…However, combined approaches including enzyme biochemistry, determination of tissue-specific expression patterns, forward genetic screening of mutants, and targeted reverse genetics by overexpression, antisense suppression, or RNAi are required to confirm functions in lignification. Although there are more than 10 million PviUTs from switchgrass (Alamo) and switchgrass (Kanlow) (Zhang et al, 2013), and more than 300 putative ESTs have been annotated as monolignol biosynthetic genes, only a few genes (4CL, COMT, CAD, and F5H) have been functionally characterized through reverse genetics in switchgrass. We have developed a gene function prediction system based on phylogenetic analysis, transcript level pattern analysis, and qRT-PCR validation.…”

“…The one internode system had been integrated into the recently published switchgrass Gene Expression Atlas (Zhang et al, 2013) for studying transcript levels in different tissues, and the suspension cell data will be integrated into the atlas in future upgrades. On the basis of this preliminary microarray analysis (signal cutoff of 100; see Supplemental Data Set 1 online, column M), 155 candidates were selected for further analysis.…”

“…We first annotated all the available PviUTs that may be involved in phenylpropanoid/ monolignol biosynthesis (Zhang et al, 2013). The PviUTs were analyzed by BlastX against the Tair10 Arabidopsis thaliana protein sequences (www.arabidopsis.org), and 331 candidates showed high sequence identity to the functionally characterized and annotated (http://cellwall.genomics.purdue.edu/index.html) monolignol pathway enzymes L-phenylalanine ammonia-lyase (PAL), 4-coumaroyl shikimate 39-hydroxylase (C39H), cinnamate 4-hydroxylase (C4H), ferulate 5-hydroxylase (F5H), 4CL, CCR, hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), COMT, or CAD (see Supplemental Data Set 1 online).…”

“…These include linkage maps constructed using a full-sib population of 238 plants and simple sequence repeat (SSR) and sequence tagged site (STS) markers (Okada et al, 2010); 61,585 high-quality ESTs from switchgrass Kanlow (Tobias et al, 2008); a collection of more than 10 million ESTs from switchgrass Alamo and switchgrass Kanlow (putative switchgrass unique transcript sequences [PviUTs]) (Zhang et al, 2013); a small EST collection from laser capture-microdissected vascular tissues (Srivastava et al, 2010); and an Affymetrix microarray chip with 122,400 probe sets representing ;90% of the transcriptome and 10-to 15-fold coverage of the genome (Zhang et al, 2013).…”

Senescence, dormancy and tillering in perennial C4 grasses

“…These studies served as a bridge to the current sequencing and annotation of the switchgrass genome available at www.phytozome.org [114], the use of microarrays [115,116], and next generation sequencing [42,117] for understanding different aspects of plant development and responses to the environment. Parallel work on the genetics and genomics of switchgrass has resulted in a deeper understanding of genome organization, linkage groups, single nucleotide polymorphisms (SNPs) and molecular markers associated with specific linkage groups, for example: [118][119][120].…”