Stephanie L. Greene

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

Economically, legumes (Fabaceae) represent the second most important family of crop plants after the grass family, Poaceae. Grain legumes account for 27% of world crop production and provide 33% of the dietary protein consumed by humans, while pasture and forage legumes provide vital part of animal feed. Fabaceae, the third largest family of flowering plants, has traditionally been divided into the following three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae, all together with 800 genera and 20,000 species. The latter subfamily contains most of the major cultivated food and feed crops. Among the grain legumes are some of mankinds earliest crop plants, whose domestication parallelled that of cereals: Soybean in China; faba bean, lentil, chickpea and pea in the Fertile Crescent of the Near East; cowpeas and bambara groundnut in Africa; soybean and mungbeans in East Asia; pigeonpea and the grams in South Asia; and common bean, lima bean, scarlet runner bean, tepary bean and lupin in Central and South America. The importance of legumes is evidenced by their high representation in ex situ germplasm collections, with more than 1,000,000 accessions worldwide. A detailed knowledge of the phylogenetic relationships of the Fabaceae is essential for understanding the origin and diversification of this economically and ecologically important family of angiosperms. This review aims to combine the phylogenetic and genetic diversity approaches to better illustrate the origin, domestication history and preserved germplasm of major legume crops from 13 genera of six tribes and to indicate further potential both for science and agriculture.

Relating Morphologic and RAPD Marker Varlation to Collection Site Environment in wild Populations of Red Clover (Trifolium Pratense L.)

Although genotypic and phenotypic markers are used to describe genetic diversity, describing patterns of variationattributable to geographic differentiation is complex.We examined concordance between morphologic and RAPDmarker classification of 33 wild red clover populations collected from the Caucasus Mountains, Russia andcompared how morphologic and RAPD markers differed in their correspondence to collection site attributes.Wealso examined if wild red clover populations collected from sites located in areas more conducive to gene flow (i.e.adjacent to roads, or drainage systems) had the same concordance between morphologic and RAPD markers aspopulations collected from sites less conducive to gene flow.We measured 15 morphologic traits in a commongarden and carried out a Random Amplified Polymorphic DNA (RAPD) analysis. There was a significantdifference among the 33 populations for 14 out of 15 morphological traits. Morphology clustered the populationsinto classes that corresponded to three climate regimes. Classification schemes generated by morphologic andRAPD data did not coincide. Morphologic data corresponded with site data for populations collected at all sites.RAPD data corresponded to site data for only those populations collected at sites not conducive to gene flow. Apopulations adaptation to collection site needs to be considered in using neutral markers to effectivelydiscriminating geographic differentiation.We discuss the practical lessons of this study on the effective collection,conservation and use of plant genetic resources.

WebGRMS: Prototype software for web-based mapping of biological collections

Biological collections are gaining recognition as priceless sources of information about the historic distribution and diversity of life. The Internet is emerging as the major venue for sharing biodiversity information since it supports globalization and broad-scale interoperability. This research demonstrates how a Web-based mapping application for biological collections was developed using WebGD, an open-source software development tool, and illustrates how simple spatial analysis help collection users describe the range of ecogeographic variation in collections and customize the selection of accessions based on georeferenced variables. Our prototype can be viewed at http://www.yukon.een.orst.edu/ greene/ms_apps/home/index.htm. The demonstration site has three functional areas: (i) Query, (ii) Analyze Collections, and (iii) Add Data. The application was developed relatively quickly and at a low cost, since the complex workings for delivering GIS functions over the Web were an internal part of the WebGD framework. Because it was based on open-source code, costs were greatly decreased compared to commercially available software. In its current form, the prototype WebGRMS application provides users interested in Medicago and Trifolium germplasm with an innovative method to better understand the germplasm collections. More importantly, we hope the prototype provides a glimpse into the future of Web-based spatial analysis of biological collections.

Occurrence of Transgenic Feral Alfalfa (Medicago sativa subsp. sativa L.) in Alfalfa Seed Production Areas in the United States.

The potential environmental risks of transgene exposure are not clear for alfalfa (Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by insects. We gathered data on feral alfalfa in major alfalfa seed-production areas in the western United States to (1) evaluate evidence that feral transgenic plants spread transgenes and (2) determine environmental and agricultural production factors influencing the location of feral alfalfa, especially transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of 4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven percent of these sites had transgenic plants. The frequency of sites having transgenic feral plants varied among our study areas. Transgenic plants were found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla Walla, respectively. Spatial analysis suggested that feral populations started independently and tended to cluster in seed and hay production areas, places where seed tended to drop. Significant but low spatial auto correlation suggested that in some instances, plants colonized nearby locations. Neighboring feral plants were frequently within pollinator foraging range; however, further research is needed to confirm transgene flow. Locations of feral plant clusters were not well predicted by environmental and production variables. However, the likelihood of seed spillage during production and transport had predictive value in explaining the occurrence of transgenic feral populations. Our study confirms that genetically engineered alfalfa has dispersed into the environment, and suggests that minimizing seed spillage and eradicating feral alfalfa along road sides would be effective strategies to minimize transgene dispersal.

Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Purpose Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer. Methods Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms. Results Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells. Conclusions CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

Genetic diversity and phenotypic variation for drought resistance in alfalfa (Medicago sativa L.) germplasm collected for drought tolerance

Drought is a major environmental factor hampering alfalfa productivity worldwide. Gene banks provide an array of trait diversity, frequently consisting of specific seed collection projects that focused on acquiring germplasm adapted to specific traits such as drought tolerance. These subsets provide a logical starting point to identify useful breeding resources. In the present study, we investigated the genetic diversity and evaluated 18 agronomic, physiological and quality traits associated with drought tolerance in a subset of 198 alfalfa accessions collected for their potential drought resistance. Significant differences were found among accessions for most of the traits evaluated. Water deficit significantly decreased biomass dry yield by 61.9% compared with well-watered control. A positive correlation was found between relative water content (RWC) and drought susceptible index, while negative correlations were found between RWC and leaf senescence (LS), and between RWC and canopy temperature (CT). Alfalfa accessions with high RWC showed relative lower yield reduction, cooler CT and delayed LS. Genetic variation for forage quality was also investigated and the correlation between agronomic and quality traits was analyzed. Biomass yield under drought was negatively correlated with total protein, relative feed value and relative forage quality, suggesting that the efforts to improve yield under drought tend to negatively affect forage quality. Therefore, maintaining forage quality should be considered for breeding alfalfa with drought resistance. Alfalfa accessions with high drought resistance scores and minimum loss of quality were identified in this study and can be used as genetic resources for developing alfalfa varieties with improved drought resistance and water use efficiency while maintaining forage quality.

Effect of Synthetic Auxin Herbicides on Seed Development and Viability in Genetically Engineered Glyphosate-Resistant Alfalfa

Feral populations of cultivated crops have the potential to function as bridges and reservoirs that contribute to the unwanted movement of novel genetically engineered (GE) traits. Recognizing that feral alfalfa has the potential to lower genetic purity in alfalfa seed production fields when it is growing in the vicinity of foraging pollinators in alfalfa seed fields, industry has established production standards to control feral plants. However, with the commercialization of GE glyphosate-resistant (GR) alfalfa and the need to support the coexistence of both GE and conventional production, effective methods to control transgenic feral alfalfa need to be developed. Therefore, a study was conducted in 2012, 2013, and 2014 to determine the effect of several synthetic auxin herbicides on seed development in GR alfalfa. GR alfalfa, var. Genuity (R44BD16), was treated with dicamba, 2,4-D, triclopyr, and aminopyralid when alfalfa plants contained green seed pods. Two weeks after herbicide application, plants were harvested, air dried, and seed yield, seed germination, and seedling emergence from the soil were determined. In 2013, dicamba, triclopyr, and 2,4-D decreased alfalfa seed yield per plant compared wih nontreated plants, whereas in 2014, all four herbicides decreased alfalfa seed yield per plant 24 to 49% (by weight) compared with nontreated plants. The same trend was evident in 2012, but seed yield was variable and was not significantly different among treatments. Seed germination averaged 43, 50, and 72% in 2012, 2013, and 2014, respectively, and was not affected by the four herbicides applied at early pod-fill stage. However, seeds harvested from plants treated with dicamba, 2,4-D, and triclopyr often produced deformed and abnormal seedlings, and when planted in soil, frequently failed to emerge. The combined effects of dicamba, 2,4-D, and triclopyr in reducing seed yield, seedling emergence, and seedling growth could contribute to managing feral alfalfa populations. Nomenclature: Aminopyralid; 2,4-D; dicamba; triclopyr; alfalfa, Medicago sativa subsp. sativa L.

Migratory Bee Hive Transportation Contributes Insignificantly to Transgenic Pollen Movement Between Spatially Isolated Alfalfa Seed Fields

Abstract Contracted commercial beekeeping operations provide an essential pollination service to many agricultural systems worldwide. Increased use of genetically engineered crops in agriculture has raised concerns over pollinator-mediated gene flow between transgenic and conventional agricultural varieties. This study evaluated whether contracted migratory beekeeping practices influence transgenic pollen flow among spatially isolated alfalfa fields. Twelve honey bee (Apis mellifera L.) colonies were permitted to forage on transgenic alfalfa blossoms for 1 wk in Touchet, WA. The hives were then transported 112 km to caged conventional alfalfa plots following one and two nights of isolation (8 and 32 h, respectively) from the transgenic source. Alfalfa seed harvested from the conventional plots was assessed for the presence of the transgene using a new seedling germination assay. We found that 8 h of isolation from a transgenic alfalfa source virtually eliminated the incidence of cross-pollination between the two varieties.

Phenotypic changes and DNA methylation status in cryopreserved seeds of rye (Secale cereale L.)

Conserving genetic diversity is a major priority of the National Laboratory for Genetic Resources Preservation (NLGRP), operated by the U.S. Department of Agriculture, Agricultural Research Service. There are two long-term preservation methods employed in the NLGRP: storage in a -18 °C freezer (conventional storage) and storage in liquid nitrogen vapor phase at -135 to -180 °C (cryopreservation). To test the phenotypic and epigenetic effects of long-term cryopreservation of orthodox seeds, we evaluated 40 cereal rye accessions (20 spring habit and 20 winter habit) stored for 25 years under both conventional storage and cryogenic conditions. In laboratory evaluations of winter habit rye, seeds from cryopreserved samples had significantly higher normal germination percentage (P < 0.05) and lower abnormal germination percentage (P < 0.05) than those stored under conventional conditions. Cryopreserved spring habit rye also had higher normal germination percentage (P < 0.01) than conventionally stored samples. In addition, winter rye seedlings from cryopreserved seeds had longer roots and smaller root diameter (P < 0.05) than seedlings from conventionally stored seeds. In field evaluations conducted in Fort Collins, Colorado in 2014-15, spikes of plants grown from conventionally stored seeds of the winter accessions were slightly longer than those from cryopreserved seeds (P = 0.045). To detect DNA methylation changes, a methylation-sensitive amplified fragment length polymorphism (metAFLP) technique was applied to two accessions. After false discovery rate adjustment, no differences in methylation were detected between storage treatments on an individual locus basis. Our study indicated that cryopreservation slowed seed deterioration as evidenced by higher germination rates compared to conventional storage, had only minimal effects on other phenotypic traits, and had no significant effects on DNA methylation status.

Abstract A35: Single cell genomic profiling of circulating tumor cells (CTCs) from metastatic colorectal cancer (mCRC) identify tumor heterogeneity and rare somatic driver alterations

Background: Mostly asymptomatic until late stage, colorectal cancer is driven by the successive accumulation of genetic alterations resulting in genomic instability within subclonal tumor populations. mCRC often progresses as a subclonally diverse multifocal disease due to selective therapeutic pressure, the surrounding tumor microenvironment, and underlying genomic heterogeneity. Targeted therapies against EGFR, VEGF or BRAF have shown increased response in a subset of patients; however, patient stratification using standard population analysis of DNA markers from tumor biopsy, (i.e. chromosomal instability, microsatellite instability, promoter methylation, resistance mutations), is problematic due to tumor heterogeneity. CTCs reflect the active metastatic subclonal populations at any given time, making single cell analysis of DNA markers a more accurate, real-time picture of cumulative metastatic diversity. Using Epic9s enrichment-free CTC analysis platform, we characterized individual CTCs from a mCRC patient to understand the extent of intra-patient genomic heterogeneity, including the presence genomic instability and point mutations. We compared the prevalence of clinically relevant subclonal alterations within patient CTCs to CRC TCGA data, offering insights into identification of therapeutic opportunities and potential mechanisms of resistance. Methods: Blood was collected from a heavily pretreated mCRC patient and was processed for CTC analysis using the Epic Platform. 34 CTCs were individually recovered, lysed, whole genome amplified, constructed into shotgun libraries and target enriched for all coding regions of 500 pan-cancer genes. Enriched libraries were sequenced to an average depth of 697X coverage by 2×150 PE sequencing. Sequences were aligned and somatic mutations were determined using VarScan with the patient9s WBC as germline reference. Variants were filtered for functional gain- or loss-of-function mutations by SIFT/PolyPhen2 and selected based on low frequency in 1000g database. Genomic instability and loss of heterozygosity (LOH) was also assessed. Somatic variants deriving from the patient CTC cohort and TCGA CRC cohort of 302 patients were annotated, analyzed, and compared using GenePool™ software (Station X). Results: MLL3 alterations, frequently observed in primary CRC biopsies (14%), were identified in 70% of all CTCs sequenced. Previously cited somatic variants were detected in minor subclonal populations of CTCs, including APC (12%), BRCA1/2 (8%), KRAS (6%), PI3KCA (6%) and TP53 (6%). A wide range of genomic instabilities and LOH was also observed across CTCs. Conclusions: The Epic CTC platform is suited to identify subclonal population of CTCs harboring clinically relevant genomic alterations on a single cell level, which can inform clonal drift, identify rare clonal populations, and enable patient stratification at higher resolution. Citation Format: Stephanie Greene, Jerry Lee, Mark Landers, Sandeep Sanga, Adam Jendrisak, Ryon Graf, Jessica Louw, Shannon Werner, Yipeng Wang, Ryan Dittamore, Dena Marrinucci. Single cell genomic profiling of circulating tumor cells (CTCs) from metastatic colorectal cancer (mCRC) identify tumor heterogeneity and rare somatic driver alterations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A35.