The Effect of Selenium Hyperaccumulation on the Astragalus Microbiome Conrad Kurowski; Grillo Lab Loyola University Chicago, Department of Biology Figure 3. Observed morphological differences in A. INTRODUCTION MATERIALS EXPERIMENTAL DESIGN crotalariae and A. lentiginosus following treatment. Astragalus is a diverse genus of Astragalus crotalariae and lentiginosus plants were plants in the Legume family Fabaceae. It grown in the greenhouse in individual 1.5” x 8.25” cone- is the largest genus of plants in terms of tainers. Four selenium treatment groups were described species and is native to designated for both species. For each treatment group, temperate regions of the Northern seven replicates of each Astragalus species was grown. Hemisphere. Legumes are perhaps most Selenate was selected because it is the main form of well-known for their symbiotic bioavailable Se in soil3. relationships with nitrogen fixating bacteria Rhizobium, and some species of • Control (C) Astragalus have gained recent attention • Selenium low (SL) –20 μM Na₂SeO₄ (5 mL) applied 1 for their production of toxic swainsonine . twice weekly Beyond this, other species of Astragalus • Selenium medium (SM) – 50 μM Na₂SeO₄ (5 mL) have been known to tolerate heavy metal applied twice weekly; soil content through hyperaccumulation. Figure 1. Sampling Locations near the Anza-Borrego • Selenium medium-high (SMH) – 80 μM Na₂SeO₄ (5 It is hypothesized that bacterial and Desert State Park in Southern California. fungal endophytes play a role in the mL) applied twice weekly; • Selenium high (SH) – 100 μM Na₂SeO₄ (5mL) applied tolerance and hyperaccumulation of Label Name Latitude Longitude Species Type [Se] (μg/g) 2 twice weekly. heavy metals in Astragalus . 1 CK1 33.275183 -115.9669 Crotalariae Soil <2.0 2 CK2 33.277633 -115.88333 Crotalariae Soil <2.0 Seeds were surface sterilized, scarified, and ABSTRACT 3 CK3 33.29685 -116.2765 Lentiginosus Soil <2.0 4 MS 19-12H 33.177867 -116.16113 Crotalariae Seed - germinated in the incubator. Following germination, seeds were transferred to cone-tainers and grown in Astragalus crotalariae grows natively 5 MS 19-14C 33.301583 -116.26648 Lentiginosus Seed - in the western United States and is known Table 1. Seed and soil samples collected for the growth field-collected soil. Cone-tainers were randomized into for its ability to hyperaccumulate heavy experiment; [Se] by ICP-AES analysis. racks for each treatment group, and racks were metal selenium in soil. Astragalus rearranged regularly in the greenhouse throughout the duration of the growth experiment. lentiginosus is a naturally co-occurring Seeds Plants were grown to an age of 4 weeks prior to non-hyperaccumulating species. These A. crotalariae and A. lentiginosus seed samples treatment. After 5 weeks of treatment, one leaflet was BIOINFORMATIC ANALYSIS organisms serve as a strong model to were collected by Matthew Scott in May of 2019 harvested from a representative A. crotalariae plant in investigate the potential role of bacterial near Ocotillo Wells, CA (MS 19-12H and MS 19-14C, both the control and selenium high groups. Leaf samples Computational Analysis communities in selenium respectively). Samples containing sufficient seeds Demultiplexed sequencing data will be trimmed were sent to the University of Illinois for ICP-MS hyperaccumulation in this system. from a single parental plant were selected. Using using CutAdapt4 to remove primer sequences. Then, quantification of elemental selenium (Figure 2). Through a manipulative greenhouse seeds from one parental reduces confounding the Dada25 pipeline will be used to classify microbial growth experiment and 16S amplicon factors such as phenotypic and genotypic variability. taxa from sequencing data. Parameters for sequencing and classification, we propose METHODS computational analysis are largely defined by the to study changes in bacterial community Soil quality of the amplicon sequencing reads generated. structure within and between Soil samples for the growth experiment were Harvest Standard operational procedures for Dada2 16s hyperaccumulating and non- collected by Dr. Grillo near Salton City, CA (CK1-3). Following validation of Selenium uptake by ICP-MS, classification will be used as a reference for our hyperaccumulating species of Astragalus Soils were subsequently subsampled and sent out for treatment groups were selected for harvesting and DNA analysis6. Classification of 16s sequences will be following treatment with sodium selenate ICP-AES quantification of elemental Selenium. This extraction. For A. crotalariae, the 0, 20, and 100 μM performed utilizing the Silva Reference Database 7. (Na₂SeO₄). analytical chemistry method allows for the treatment groups were selected. For A. lentiginosus v. Amplicon sequencing variant (ASV) and taxonomy quantification of selenium content in soil samples. borreganus, the 0, 20, and 80 μM treatment groups tables generated from the Dada2 pipeline will be REFERENCES Our measured concentrations of <2.0 μg/g border were selected. For each plant, four sampling locations processed downstream in R studio. 1. Cook D, Gardner DR, Grum D, et al. Swainsonine and endophyte the limit of Selenium deficient areas. were used: rhizosphere, root, whole soil, and leaf tissue. relationships in Astragalus mollissimus and Astragalus lentiginosus. J Agric Food Chem. 2011;59(4):1281-1287. doi:10.1021/Jf103551t Statistical Analyses 2. Sura-de Jong M, Reynolds RJB, Richterova K, et al. Selenium DNA Extraction Alpha diversity, including species richness, hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth Following the completion of harvesting protocol, evenness, and relative abundance, will be calculated Crotalaire Leaf Tissue [Se] by ICP-MS promoting properties. Front Plant Sci. 2015;6. leaf, root, rhizosphere, and whole soil samples were for samples. Analysis will be performed in R Studio doi:10.3389/fpls.2015.00113 (ppm) 3. Prins CN, Hantzis LJ, Valdez-Barillas JR, et al. Getting to the Root of transferred to DNEasy Powersoil DNA Isolation Kit bead- utilizing the packages Phyloseq9 and Biostrings10. Selenium Hyperaccumulation—Localization and Speciation of Root Selenium and Its Effects on Nematodes. Soil Syst. 2019;3(3):47. beating tubes. DNA isolation was performed following Microbiome Analyst8, a web-based platform for doi:10.3390/soilsystems3030047 4. Martin M. Cutadapt removes adapter sequences from high- Qiagen’s protocol, with a few modifications. Optional visualization of amplicon sequencing data, will also 100 µM Na2Se04 417 throughput sequencing reads. EMBnet.journal. 2011;17(1):10-12. incubation at 60℃ for 10 minutes prior to bead beating doi:10.14806/ej.17.1.200 CK100 be utilized. 5. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, was performed. Fifty microliters of elution buffer was Beta diversity calculations will be performed for Holmes SP. DADA2: High resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581-583. used to elute extracted DNA, and spin columns were comparisons between samples. Unweighted-UniFrac doi:10.1038/nmeth.3869 incubated with elution buffer for 10 minutes at 30℃ distances, Bray-Curtis Similarity, or Jaccard Similarity 6. DADA2 Pipeline Tutorial (1.12). http://benjJneb.github.io/dada2/tutorial.html. Accessed October 9, prior to final elution. will be used to generateREFERENCES distance matrices between 2019. 0 µM Na2Se04 46.7 7. The SILVA ribosomal RNA gene database proJect: improved data CK00 different sample pairs. Then, multiple distance processingCONTACT and web-based tools. 16S Amplification and SeQuencing matrices1. Click here will to beinsert combined your References. and used Type itfor in orordinations copy and paste https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531112/. Accessed from your Word document or other source. January 29, 2020. Extracted DNA samples were handed off to the such as PCoA or NMDS. Linear Discriminant Analysis 8. Dhariwal A, Chong J, Habib S, King IL, Agellon LB, Xia J. 0 100 200 300 400 500 2. Click on the border once to highlight and select a different font MicrobiomeAnalyst: a web-based tool for comprehensive statistical, Loyola Genomics Facility for 16S PCR amplification. and Analysis of Effect Size will be performed to or font size that suits you. This text is in Calibri 28pt and is easily visual and meta-analysis of microbiome data. Nucleic Acids Res. Amplification of the 16S V4 hypervariable region will be identify significantly different taxa between 2017;45(W1):W180-W188. doi:10.1093/nar/gkx295 Figure 2. Selenium quantification of one leaflet in readable up to 4 feet away. 9. phyloseq: An R Package for Reproducible Interactive Analysis and performed using the primer set 515F/806R. Following Astragalus species and between different treatment Graphics of Microbiome Census Data. representative A. crotolariae plants, following 5 weeks of amplifications, 2x250 paired end sequencing will be https://Journals.plos.org/plosone/article?id=10.1371/journal.pone.006 treatment. groups. 1217. Accessed January 29, 2020. performed on an Illumina MiSeq platform. 10. Pagès H, Aboyoun P, Gentl eman R , DebRoy S. Biostrings: Efficient Manipulation of Biological Strings. Bioconductor version: Release (3.10); 2020. doi:10.18129/B9.bioc.Biostrings.