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Quinoa (Chenopodium quinoa Willd.) is an important food crop for subsistence farmers in the Altiplano (high plains) of Peru, Bolivia, and Argentina. Saponins are part of a diverse family of secondary metabolites that are found in high concentrations in the pericarp of many varieties of quinoa. Due to their bitter taste and anti-nutritive properties, saponins must be removed before the quinoa grain is consumed. There are ‘sweet’ varieties of quinoa that have significantly reduced levels of saponin. Previous research suggests saponin production is controlled by a single locus. The major objective of this research was to elucidate the genetic components in the saponin biosynthesis pathway. Thus, we report the development and annotation of the first large scale expressed sequence tag (EST) collection for quinoa based on Sanger and 454 pyrosequencing of maturing seed tissue expressing saponins. Sanger sequencing produced 18,325 reads with an average read length of 693 nucleotides, while 454 GS-FLX pyrosequencing generated 295,048 reads with an average read length of 202 nucleotides. A hybrid assembly of all sequences generated 39,366 unigenes, consisting of 16,728 contigs and 22,638 singletons. Repeat sequence analysis of the unigene set identified 291 new microsatellite markers. From the unigene set, a custom microarray was developed and used to assay transcriptional changes in developing seeds of saponin-containing and saponin-free quinoa lines. The microarray consisted of 102,834 oligonucleotide probes representing 37,716 sequences of the unigenes set. Three different statistical comparisons, based on comparisons of ‘sweet’ vs. ‘bitter’ seed tissue at two developmental stages, were assayed on the custom array. Using a p-value cutoff threshold of 0.01, we identified a list of 198 significantly differentially expressed candidate genes common to all three comparisons. We also identified a list of candidate genes (p-value ≤ 0.05) that are known to be associated with identified triterpenoid (saponin) biosynthetic pathways that were differentially expressed in all three comparisons. Included in this list are candidate genes that share homology to cytochrome P450s (20), cytochrome P450 monooxygenases (10), and glycosyltransferases (49) suggesting that transcriptional differences in the saponin biosynthesis pathway possibly responsible for the absence or presence of saponin in quinoa are determined after the formation of the β-amyrin skeleton. These candidate genes are suggested for use in future studies in the production of saponin in quinoa.

Big sagebrush (Artemisia tridentata) is one of the ecologically most important shrub species in western North America. The species serves as a major source of food and habitat for the near-threatened sage grouse and various other fauna. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is considered as a serious threat to sustainability of the big sagebrush ecosystem. Because of its importance, restoration of this species is very crucial to those dependent on big sagebrush community. However, restoration of big sagebrush carried out by using diverse seed source can lead to imbalance and degradation in the native ecosystem. Therefore, restoration works aided by understanding of adaptive traits of big sagebrush using molecular markers will aid successful restoration. The major objective of this research was to create a substantial resource of nuclear sequence data and identify markers that can be used in future studies in big sagebrush. We report the development and annotation of the first expressed sequence tag (EST) collection for big sagebrush based on 454 sequencing of leaf tissue. Expressed genes of subspecies tridentata and vaseyana were sequenced using the 454 GS-FLX titanium platform, which produced 823,392 reads with an average read length of 404 bp and 702,001 reads with an average read length of 333 bp for sspp. tridentata and vaseyana, respectively. Assembly of the reads resulted in 212,102 consensus sequences in ssp. tridentata and 199,439 in ssp. vaseyana. A combined assembly of both subspecies sequences generated 29,541 contigs with an average length of 796 bp and 275,866 singletons with an average length of 370 bp. A BLASTx search against the non-redundant (NR) protein database using the contigs obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e-15). Gene Ontology (GO) IDs were assigned to 18,397 sequences. A total of 20,952 SNPs were detected between the two subspecies and 1,182 SNPs were confirmed in tetraploid ssp. wyomingensis. In addition, 1,003 and 507 SSRs were detected in ssp. tridentata contigs and ssp. vaseyana contigs, respectively.