The genetics of Ug99 stem rust resistance in spring wheat variety 'Linkert'.

Wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt) threatens wheat production worldwide. The objective of this study was to characterize wheat stem rust resistance in 'Linkert', a variety with adult plant resistance effective to emerging wheat stem rust pathogen strain Ug99. Two doubled haploid (DH) populations and one recombinant inbred line (RIL) population were developed with 'Linkert' as a stem rust resistant parent. Hard red spring wheat variety 'Forefront' and genetic stock 'LMPG' were used as stem rust susceptible parents of the DH populations. Breeding line 'MN07098-6' was used as a susceptible parent of the RIL population. Both DH and RIL populations with their parents were evaluated both at the seedling stage and in the field against Pgt races. Genotyping data of the DH populations were generated using the wheat iSelect 90k SNP assay. The RIL population was genotyped by genotyping-by-sequencing. We found QTL consistently associated with wheat stem rust resistance on chromosome 2BS for the Linkert/Forefront DH population and the Linkert/MN07098-6 RIL population both in Ethiopia and Kenya. Additional reliable QTL were detected on chromosomes 5BL (125.91 cM) and 4AL (Sr7a) for the Linkert/LMPG population in Ethiopia and Kenya. Different QTL identified in the populations reflect the importance of examining the genetics of resistance in populations derived from adapted germplasm (Forefront and MN07098-6) in addition to a genetic stock (LMPG). The associated markers in this study could be used to track and select for the identified QTL in wheat breeding programs.

Developing adapted wheat lines with broad-spectrum resistance to stem rust: Introgression of Sr59 through backcrossing and selections based on genotyping-by-sequencing data.

Control of stem rust, caused by Puccinia graminis f.sp. tritici, a highly destructive fungal disease of wheat, faces continuous challenges from emergence of new virulent races across wheat-growing continents. Using combinations of broad-spectrum resistance genes could impart durable stem rust resistance. This study attempted transfer of Sr59 resistance gene from line TA5094 (developed through CSph1bM-induced T2DS·2RL Robertsonian translocation conferring broad-spectrum resistance). Poor agronomic performance of line TA5094 necessitates Sr59 transfer to adapted genetic backgrounds and utility evaluations for wheat improvement. Based on combined stem rust seedling and molecular analyses, 2070 BC1F1 and 1230 BC2F1 plants were derived from backcrossing BAJ#1, KACHU#1, and REEDLING#1 with TA5094. Genotyping-by-sequencing (GBS) results revealed the physical positions of 15,116 SNPs on chromosome 2R. The adapted genotypes used for backcrossing were found not to possess broad-spectrum resistance to selected stem rust races, whereas Sr59-containing line TA5094 showed resistance to all races tested. Stem rust seedling assays combined with kompetitive allele-specific PCR (KASP) marker analysis successfully selected and generated the BC2F2 population, which contained the Sr59 gene, as confirmed by GBS. Early-generation data from backcrossing suggested deviations from the 3:1 segregation, suggesting that multiple genes may contribute to Sr59 resistance reactions. Using GBS marker data (40,584 SNPs in wheat chromosomes) to transfer the recurrent parent background to later-generation populations resulted in average genome recovery of 71.2% in BAJ#1*2/TA5094, 69.8% in KACHU#1*2/TA5094, and 70.5% in REEDLING#1*2/TA5094 populations. GBS data verified stable Sr59 introgression in BC2F2 populations, as evidenced by presence of the Ph1 locus and absence of the 50,936,209 bp deletion in CSph1bM. Combining phenotypic selections, stem rust seedling assays, KASP markers, and GBS data substantially accelerated transfer of broad-spectrum resistance into adapted genotypes. Thus, this study demonstrated that the Sr59 resistance gene can be introduced into elite genetic backgrounds to mitigate stem rust-related yield losses.

Discussion: Prioritize perennial grain development for sustainable food production and environmental benefits.

Perennial grains have potential to contribute to ecological intensification of food production by enabling the direct harvest of human-edible crops without requiring annual cycles of disturbance and replanting. Studies of prototype perennial grains and other herbaceous perennials point to the ability of agroecosystems including these crops to protect water quality, enhance wildlife habitat, build soil quality, and sequester soil carbon. However, genetic improvement of perennial grain candidates has been hindered by limited investment due to uncertainty about whether the approach is viable. As efforts to develop perennial grain crops have expanded in past decades, critiques of the approach have arisen. With a recent report of perennial rice producing yields equivalent to those of annual rice over eight consecutive harvests, many theoretical concerns have been alleviated. Some valid questions remain over the timeline for new crop development, but we argue these may be mitigated by implementation of recent technological advances in crop breeding and genetics such as low-cost genotyping, genomic selection, and genome editing. With aggressive research investment in the development of new perennial grain crops, they can be developed and deployed to provide atmospheric greenhouse gas reductions.

Identification of a Small Translocation from 6R Possessing Stripe Rust Resistance to Wheat.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Eriks. & E. Henn, is the most devastating fungal disease of bread wheat. Here, a wheat-rye multiple disomic substitution line, SLU126 4R (4D), 5R (5D), and 6R (7D), possessing resistance against 25 races of P. striiformis f. sp. tritici, was used and crossed with Chinese Spring ph1b to induce homeologous recombination to produce introgressions with a reduced rye chromosome segment. Seedling assays confirmed that the stripe rust resistance from SLU126 was retained over multiple generations. Through genotyping-by-sequencing (GBS) platforms and aligning the putative GBS-single-nucleotide polymorphism (SNPs) to the full-length annotated rye nucleotide-binding leucine-rich repeat (NLR) genes in the parental lines (CS ph1b, SLU126, CSA, and SLU820), we identified the physical position of 26, 13, and 9 NLR genes on chromosomes 6R, 4R, and 5R, respectively. The physical positions of 25 NLR genes on chromosome 6R were identified from 568,460,437 bp to 879,958,268 bp in the 6RL chromosome segment. Based on these NLR positions on the 6RL chromosome segment, the three linked SNPs (868,123,650 to 873,285,112 bp) were validated through kompetitive allele-specific PCR (KASP) assays in SLU126 and resistance plants in the family 29-N3-5. Using these KASP markers, we identified a small piece of the rye translocation (i.e., as a possible 6DS.6DL.6RL.6DL) containing the stripe resistance gene, temporary designated YrSLU, within the 6RL segment. This new stripe rust resistance gene provides an additional asset for wheat improvement to mitigate yield losses caused by stripe rust.

Genome-wide association mapping for field and seedling resistance to the emerging Puccinia graminis f. sp. tritici race TTRTF in wheat.

Stem rust of wheat (Triticum spp.), caused by Puccinia graminis f. sp. tritici (Pgt), is one of the most impactful wheat diseases because of its threat to global wheat production. While disease mitigation has primarily been achieved through the deployment of resistant wheat varieties, emerging new virulent races continue to pose risks to the crop. For example, races such as Ug99 (TTKSK), TKTTF, and TTRTF have caused epidemics in different wheat growing regions of the world in recent years. A continual search for new and effective sources of resistance is therefore necessary to safeguard wheat production. This study assessed a breeding panel from the Ethiopian Institute of Agricultural Research (EIAR) wheat breeding program for seedling and field plant resistance to TTRTF and reports genomic regions conferring resistance to TTRTF. Trait correlations (r) were medium to strong (range = .38-.71) and heritabilities were moderate (.32-.56). Association analysis for resistance to TTRTF resulted in detection of 20 markers in 11 chromosomes; the marker S1B_175439851 was associated with resistance at both seedling and adult plant stages. Models with two to four QTL combinations reduced seedling and field disease severity by 12-48 and 9-17%, respectively. Genomic prediction for TTRTF resistance resulted in low to moderately-high predictions (mean correlations of .25-.47). Identification of resistant lines and QTL in the EIAR population is expected to assist in selection toward improved resistance to TTRTF. Specifically, the application of genomic selection (GS) in identifying resistant lines in future related breeding populations will further assist breeding efforts against this new stem rust pathogen race.

Influence of Pollen Dispersal and Mating Pattern in Domestication of Intermediate Wheatgrass, a Novel Perennial Food Crop.

Intermediate wheatgrass (IWG) is a perennial forage grass that is currently being domesticated as a grain crop. It is a primarily wind-pollinated outcrossing species and expresses severe inbreeding depression when self-pollinated. Characterization of pollen dispersal, mating parameters, and change in genetic diversity due to pollen movement is currently lacking in IWG. In this study, we examined pollen dispersal in an IWG selection nursery by evaluating 846 progeny from 15 mother plants and traced their parentage to 374 fathers. A set of 2,500 genomic loci was used to characterize the population. We assigned paternity to 769 (91%) progeny and the average number of fathers per mother plant was 37, from an average of 56 progeny examined per mother. An extensive number (80%) of pollination events occurred within 10 m of the mother plants. Pollination success was not correlated with trait attributes of the paternal genotypes. Mating system analysis confirmed that IWG is highly outcrossing and inbreeding was virtually absent. Neither genetic diversity nor the genome-estimated trait values of progeny were significantly affected by pollinator distance. The distance of pollinator in an IWG breeding nursery therefore was not found to be a major contributor in maintaining genetic diversity. These findings reveal the pollen dispersal model in IWG for the first time and its effect on genetic diversity, which will be valuable in designing future IWG breeding populations. Information generated and discussed in this study could be applied in understanding gene flow and genetic diversity of other open-pollinated species.

Genome-wide association mapping and genomic prediction for kernel color traits in intermediate wheatgrass (Thinopyrum intermedium).

BACKGROUND: Intermediate wheatgrass (IWG) is a novel perennial grain crop currently undergoing domestication. It offers important ecosystem benefits while producing grain suitable for human consumption. Several aspects of plant biology and genetic control are yet to be studied in this new crop. To understand trait behavior and genetic characterization of kernel color in IWG breeding germplasm from the University of Minnesota was evaluated for the CIELAB components (L*, a*, b*) and visual differences. Trait values were used in a genome-wide association scan to reveal genomic regions controlling IWG's kernel color. The usability of genomic prediction in predicting kernel color traits was also evaluated using a four-fold cross validation method. RESULTS: A wide phenotypic variation was observed for all four kernel color traits with pairwise trait correlations ranging from - 0.85 to 0.27. Medium to high estimates of broad sense trait heritabilities were observed and ranged from 0.41 to 0.78. A genome-wide association scan with single SNP markers detected 20 significant marker-trait associations in 9 chromosomes and 23 associations in 10 chromosomes using multi-allelic haplotype blocks. Four of the 20 significant SNP markers and six of the 23 significant haplotype blocks were common between two or more traits. Evaluation of genomic prediction of kernel color traits revealed the visual score to have highest mean predictive ability (r2 = 0.53); r2 for the CIELAB traits ranged from 0.29-0.33. A search for candidate genes led to detection of seven IWG genes in strong alignment with MYB36 transcription factors from other cereal crops of the Triticeae tribe. Three of these seven IWG genes had moderate similarities with R-A1, R-B1, and R-D1, the three genes that control grain color in wheat. CONCLUSIONS: We characterized the distribution of kernel color in IWG for the first time, which revealed a broad phenotypic diversity in an elite breeding germplasm. Identification of genetic loci controlling the trait and a proof-of-concept that genomic selection might be useful in selecting genotypes of interest could help accelerate the breeding of this novel crop towards specific end-use.

Molecular Characterization of Genomic Regions for Adult Plant Resistance to Stem Rust in a Spring Wheat Mapping Population.

Adult plant resistance (APR) to wheat stem rust has been one of the approaches for resistance breeding since the evolution of the Ug99 race group and other races. This study was conducted to dissect and understand the genetic basis of APR to stem rust in spring wheat line 'Copio'. A total of 176 recombinant inbred lines (RILs) from the cross of susceptible parent 'Apav' with Copio were phenotyped for stem rust resistance in six environments. Composite interval mapping using 762 genotyping-by-sequencing markers identified 16 genomic regions conferring stem rust resistance. Assays with gene-linked molecular markers revealed that Copio carried known APR genes Sr2 and Lr46/Yr29/Sr58 in addition to the 2NS/2AS translocation that harbors race-specific genes Sr38, Lr37, and Yr17. Three quantitative trait loci (QTLs) were mapped on chromosomes 2B, two QTLs on chromosomes 3A, 3B, and 6A each, and one QTL on each of chromosomes 2A, 1B, 2D, 4B, 5D, 6D, and 7A. The QTL QSr.umn.5D is potentially a new resistance gene and contributed to quantitative resistance in Copio. The RILs with allelic combinations of Sr2, Sr38, and Sr58 had 27 to 39% less stem rust coefficient of infection in all field environments compared with RILs with none of these genes, and this gene combination was most effective in the U.S. environments. We conclude that Copio carries several genes that provide both race-specific and non-race-specific resistance to diverse races of stem rust fungus and can be used by breeding programs in pyramiding other effective genes to develop durable resistance in wheat.

Genetic architecture of agronomic and quality traits in a nested association mapping population of spring wheat.

Germplasm collections are rich sources of genetic variation to improve crops for many valuable traits. Nested association mapping (NAM) populations can overcome the limitations of genome-wide association studies (GWAS) in germplasm collections by reducing the effect of population structure. We exploited the genetic diversity of the USDA-ARS wheat (Triticum aestivum L.) core collection by developing the Spring Wheat Multiparent Introgression Population (SWMIP). To develop this population, twenty-five core parents were crossed and backcrossed to the Minnesota spring wheat cultivar RB07. The NAM population and 26 founder parents were genotyped using genotyping-by-sequencing and phenotyped for heading date, height, test weight, and grain protein content. After quality control, 20,312 markers with physical map positions were generated for 2,038 recombinant inbred lines (RILs). The number of RILs in each family varied between 58 and 96. Three GWAS models were utilized for quantitative trait loci (QTL) detection and accounted for known family stratification, genetic kinship, and both covariates. GWAS was performed on the whole population and also by bootstrap sampling of an equal number of RILs from each family. Greater power of QTL detection was achieved by treating families equally through bootstrapping. In total 16, 15, 12, and 13 marker-trait associations (MTAs) were identified for heading date, height, test weight, and grain protein content, respectively. Some of these MTAs were coincident with major genes known to control the traits, but others were novel and contributed by the wheat core parents. The SWMIP will be a valuable source of genetic variation for spring wheat breeding.

Dominance and G×E interaction effects improve genomic prediction and genetic gain in intermediate wheatgrass (Thinopyrum intermedium).

Genomic selection (GS) based recurrent selection methods were developed to accelerate the domestication of intermediate wheatgrass [IWG, Thinopyrum intermedium (Host) Barkworth & D.R. Dewey]. A subset of the breeding population phenotyped at multiple environments is used to train GS models and then predict trait values of the breeding population. In this study, we implemented several GS models that investigated the use of additive and dominance effects and G×E interaction effects to understand how they affected trait predictions in intermediate wheatgrass. We evaluated 451 genotypes from the University of Minnesota IWG breeding program for nine agronomic and domestication traits at two Minnesota locations during 2017-2018. Genet-mean based heritabilities for these traits ranged from 0.34 to 0.77. Using four-fold cross validation, we observed the highest predictive abilities (correlation of 0.67) in models that considered G×E effects. When G×E effects were fitted in GS models, trait predictions improved by 18%, 15%, 20%, and 23% for yield, spike weight, spike length, and free threshing, respectively. Genomic selection models with dominance effects showed only modest increases of up to 3% and were trait-dependent. Cross-environment predictions were better for high heritability traits such as spike length, shatter resistance, free threshing, grain weight, and seed length than traits with low heritability and large environmental variance such as spike weight, grain yield, and seed width. Our results confirm that GS can accelerate IWG domestication by increasing genetic gain per breeding cycle and assist in selection of genotypes with promise of better performance in diverse environments.

Enhancing Crop Domestication Through Genomic Selection, a Case Study of Intermediate Wheatgrass.

Perennial grains could simultaneously provide food for humans and a host of ecosystem services, including reduced erosion, minimized nitrate leaching, and increased carbon capture. Yet most of the world's food and feed is supplied by annual grains. Efforts to domesticate intermediate wheatgrass (Thinopyrumn intermedium, IWG) as a perennial grain crop have been ongoing since the 1980's. Currently, there are several breeding programs within North America and Europe working toward developing IWG into a viable crop. As new breeding efforts are established to provide a widely adapted crop, questions of how genomic and phenotypic data can be used among sites and breeding programs have emerged. Utilizing five cycles of breeding data that span 8 years and two breeding programs, University of Minnesota, St. Paul, MN, and The Land Institute, Salina, KS, we developed genomic selection (GS) models to predict IWG traits. Seven traits were evaluated with free-threshing seed, seed mass, and non-shattering being considered domestication traits while agronomic traits included spike yield, spikelets per inflorescence, plant height, and spike length. We used 6,199 genets - unique, heterozygous, individual plants - that had been profiled with genotyping-by-sequencing, resulting in 23,495 SNP markers to develop GS models. Within cycles, the predictive ability of GS was high, ranging from 0.11 to 0.97. Across-cycle predictions were generally much lower, ranging from -0.22 to 0.76. The prediction ability for domestication traits was higher than agronomic traits, with non-shattering and free threshing prediction abilities ranging from 0.27 to 0.75 whereas spike yield had prediction abilities ranging from -0.22 to 0.26. These results suggest that progress to reduce shattering and increase the percent free-threshing grain can be made irrespective of the location and breeding program. While site-specific programs may be required for agronomic traits, synergies can be achieved in rapidly improving key domestication traits for IWG. As other species are targeted for domestication, these results will aid in rapidly domesticating new crops.

Genome-Wide Association Study of Yield Component Traits in Intermediate Wheatgrass and Implications in Genomic Selection and Breeding.

Intermediate wheatgrass (Thinopyrum intermedium, IWG) is a perennial grain crop with high biomass and grain yield, long seeds, and resistance to pests and diseases. It also reduces soil erosion, nitrate and mineral leaching into underground water tables, and sequesters carbon in its roots. The domestication timeline of IWG as a grain crop spans only 3 decades, hence it lags annual grain crops in yield and seed characteristics. One approach to improve its agronomic traits is by using molecular markers to uncover marker-trait associations. In this study, we performed association mapping on IWG breeding germplasm from the third recurrent selection cycle at the University of Minnesota. The IWG population was phenotyped in St Paul, MN in 2017 and 2018, and in Crookston, MN in 2018 for grain yield, seed length, width and weight, spike length and weight, and number of spikelets per spike. Strong positive correlations were observed among most trait pairs, with correlations as high as 0.76. Genotyping using high throughput sequencing identified 8,899 high-quality genome-wide SNPs which were combined with phenotypic data in association mapping to discover regions associated with the yield component traits. We detected 154 genetic loci associated with these traits of which 19 were shared between at least two traits. Prediction of breeding values using significant loci as fixed effects in genomic selection model improved predictive abilities by up to 14%. Genetic mapping of agronomic traits followed by using genomic selection to predict breeding values can assist breeders in selecting superior genotypes to accelerate IWG domestication.

Phylogenetic analyses and in-seedling expression of ammonium and nitrate transporters in wheat.

Plants deploy several ammonium transporter (AMT) and nitrate transporter (NRT) genes to acquire NH4+ and NO3- from the soil into the roots and then transport them to other plant organs. Coding sequences of wheat genes obtained from ENSEMBL were aligned to known AMT and NRT sequences of Arabidopsis, barley, maize, rice, and wheat to retrieve homologous genes. Bayesian phylogenetic relationships among these genes showed distinct classification of sequences with significant homology to NRT1, NRT2, and NRT3 (NAR2). Inter-species gene duplication analysis showed that eight AMT and 77 NRT genes were orthologous to the AMT and NRT genes of aforementioned plant species. Expression patterns of these genes were studied via whole transcriptome sequencing of 21-day old seedlings of five spring wheat lines. Eight AMT and 52 NRT genes were differentially expressed between root and shoot; and 131 genes did not express neither in root nor in shoot of 21-day old seedlings. Homeologous genes in the A, B, and D genomes, characterized by high sequence homology, revealed that their counterparts exhibited different expression patterns. This complement and evolutionary relationship of wheat AMT and NRT genes is expected to help in development of wheat germplasm with increased efficiency in nitrogen uptake and usage.

Nested Association Mapping of Stem Rust Resistance in Wheat Using Genotyping by Sequencing.

We combined the recently developed genotyping by sequencing (GBS) method with joint mapping (also known as nested association mapping) to dissect and understand the genetic architecture controlling stem rust resistance in wheat (Triticum aestivum). Ten stem rust resistant wheat varieties were crossed to the susceptible line LMPG-6 to generate F6 recombinant inbred lines. The recombinant inbred line populations were phenotyped in Kenya, South Africa, and St. Paul, Minnesota, USA. By joint mapping of the 10 populations, we identified 59 minor and medium-effect QTL (explained phenotypic variance range of 1% - 20%) on 20 chromosomes that contributed towards adult plant resistance to North American Pgt races as well as the highly virulent Ug99 race group. Fifteen of the 59 QTL were detected in multiple environments. No epistatic relationship was detected among the QTL. While these numerous small- to medium-effect QTL are shared among the families, the founder parents were found to have different allelic effects for the QTL. Fourteen QTL identified by joint mapping were also detected in single-population mapping. As these QTL were mapped using SNP markers with known locations on the physical chromosomes, the genomic regions identified with QTL could be explored more in depth to discover candidate genes for stem rust resistance. The use of GBS-derived de novo SNPs in mapping resistance to stem rust shown in this study could be used as a model to conduct similar marker-trait association studies in other plant species.

Deep sequencing of amplicons reveals widespread intraspecific hybridization and multiple origins of polyploidy in big sagebrush (Artemisia tridentata; Asteraceae).

PREMISE OF THE STUDY: Hybridization has played an important role in the evolution and ecological adaptation of diploid and polyploid plants. Artemisia tridentata (Asteraceae) tetraploids are extremely widespread and of great ecological importance. These tetraploids are often taxonomically identified as A. tridentata subsp. wyomingensis or as autotetraploids of diploid subspecies tridentata and vaseyana. Few details are available as to how these tetraploids are formed or how they are related to diploid subspecies. • METHODS: We used amplicon sequencing to assess phylogenetic relationships among three recognized subspecies: tridentata, vaseyana, and wyomingensis. DNA sequence data from putative genes were pyrosequenced and assembled from 329 samples. Nucleotide diversity and putative haplotypes were estimated from the high-read coverage. Phylogenies were constructed from Bayesian coalescence and neighbor-net network analyses. • KEY RESULTS: Analyses support distinct diploid subspecies of tridentata and vaseyana in spite of known hybridization in ecotones. Nucleotide diversity estimates of populations compared to the total diversity indicate the relationships are predominately driven by a small proportion of the amplicons. Tetraploids, including subspecies wyomingensis, are polyphyletic occurring within and between diploid subspecies groups. • CONCLUSIONS: Artemisia tridentata is a species comprising phylogenetically distinct diploid progenitors and a tetraploid complex with varying degrees of phylogenetic and morphological affinities to the diploid subspecies. These analyses suggest tetraploids are formed locally or regionally from diploid tridentata and vaseyana populations via autotetraploidy, followed by introgression between tetraploid groups. Understanding the phylogenetic vs. ecological relationships of A. tridentata subspecies will have bearing on how to restore these desert ecosystems.

Transcriptome characterization and polymorphism detection between subspecies of big sagebrush (Artemisia tridentata).

BACKGROUND: Big sagebrush (Artemisia tridentata) is one of the most widely distributed and ecologically important shrub species in western North America. This species serves as a critical habitat and food resource for many animals and invertebrates. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is a serious threat to big sagebrush ecosystem sustainability. Lack of genomic data has limited our understanding of the evolutionary history and ecological adaptation in this species. Here, we report on the sequencing of expressed sequence tags (ESTs) and detection of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in subspecies of big sagebrush. RESULTS: cDNA of A. tridentata sspp. tridentata and vaseyana were normalized and sequenced using the 454 GS FLX Titanium pyrosequencing technology. Assembly of the reads resulted in 20,357 contig consensus sequences in ssp. tridentata and 20,250 contigs in ssp. vaseyana. A BLASTx search against the non-redundant (NR) protein database using 29,541 consensus sequences obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e⁻¹5). A total of 20,952 SNPs and 119 polymorphic SSRs were detected between the two subspecies. SNPs were validated through various methods including sequence capture. Validation of SNPs in different individuals uncovered a high level of nucleotide variation in EST sequences. EST sequences of a third, tetraploid subspecies (ssp. wyomingensis) obtained by Illumina sequencing were mapped to the consensus sequences of the combined 454 EST assembly. Approximately one-third of the SNPs between sspp. tridentata and vaseyana identified in the combined assembly were also polymorphic within the two geographically distant ssp. wyomingensis samples. CONCLUSION: We have produced a large EST dataset for Artemisia tridentata, which contains a large sample of the big sagebrush leaf transcriptome. SNP mapping among the three subspecies suggest the origin of ssp. wyomingensis via mixed ancestry. A large number of SNP and SSR markers provide the foundation for future research to address questions in big sagebrush evolution, ecological genetics, and conservation using genomic approaches.