Molecular AdaptationsOryza glaberrima of

Megan Onyundo, Andrew Disharoon, Lindsay Shields, Amy Lawton-Rauh

Introduction 416 (4.6%) Methods SeedsResults 53 (5.3%) 37 (3.7%) is a staple food around planted Nine separate Trinidad rice genotypes were provided through a collaboration with 1000 (100%) replanted Tissue Francis Morean (ethnobotanist, Trinidad), David Shields (USC), and Michael Purugga- the world providing up to nan (NYU). Additionally, 110 accessions of glaberrima were provided by the 66% of caloric intake to Seeds placed samples USDA GRIN repository (Fig. 4). C many people in developing in tubes A Collections of Trinidad rice and Oryza countries. , glaberrima were germinated using also known as African rice, Figure 5: Quantitative attrition of successful growth from seeds 12.5 mL of 10 mM calcium chloride has a independent origin solution in a 50 mL conical vial (Fig. than (Asian To determine if gene size differences in these key genes can be used to 7C). Seeds were prepared for germi- rice) and was domesticated distinguish between O. sativa and O. glaberrima genomic backgrounds, nation by removing the hull and then B in areas surrounding the we test PCR primers for each of these genes (Fig. 6). Two samples gave scarified using sandpaper (Fig. 7A,B). over 3,000 years clear results in the gel including the loci, OsDREB2A and OsBBPI. The Once placed in the vial, they were ago (Fig. 1). Oryza Figure 1: Origins of O. sativa and O. glaberrima bands sizes are equivalent in O. sativa and O. glaberrima, thus there are placed in a 28 degrees C incubator. glaberrima varieties were introduced to other countries and records sug- no insertions or deletions in that position. In contrast, the ATPb gene gest that they were crossed with O. sativa, resulting in rice resilient to only amplified in Oryza glaberrima and our first trials of HKT1 and HD1 stressful conditions and with unique properties in places such as Trinidad did not work thus we need to try again. While with the HD1 gene we Figure 7: Oryza glaberrima seeds with husks (A & and Southeast North America (South Carolina/Georgia region). Over time, should have seen amplification of the loci in Oryza sativa, which B), Oryza glaberrima seeds in CaCl2 soultion (C) rice has become embedded in Trinidad culture, yet the genomic back- means there was an error in the PCR reaction. However, we knew we The seeds were planted once the ground and mechanisms of adaptation unique to Trinidad rice are un- would not see results in Oryza glaberrima because the gene is not pres- shoot of the seed was approxi- known (Fig. 3). The growth habits of Trinidad rice are more similar to O. ent in the genome. glaberrima, thus we hypothesize that Trinidad rice has a higher proportion mately 3 cm tall (Fig. 8). Plants of the Oryza glaberrima genome. O. were repotted at first tiller stage. glaberrima has not been as thor- oughly studied as O. sativa, there- fore, further genetic analysis on O. Figure 8: Set up of planted Oryza glamberrima A glaberrima is needed to resolve the At second tiller stage (Fig. 9A), 100 unknown genetic and domestica- mg of tissue was collected (Fig. 9B) tion history of Trinidad rice. This in a 2 mL Eppendorf tube and placed investigation of African rice fo- in the -80 degrees C freezer until cused on establishing a germina- further analysis. Genomic DNA ex- tion protocol (Fig. 2) and on devel- tractions were performed the Mach- B Figure 2: Growth progression of Oryza glaberrima oping germplasm for genome erey-Nagel kit (NucleoSpin II, analyses of regions containing specific stress resilience and domestica- REF 740770.250), and then quality tion loci including: HTK1 (salt tolerance), OsDREB2A (drought tolerance), control was determined using the OsBBPI (antiherbivore defense mechanism), HD1 (flowering time), and Figure 6: Gel image of PCR results using DNA from O. sative, O. glaberrima, and negative control testing Nanodrop 800 (Thermo Scientific) to primers for amplification of HKT1, OsDREB2A, OsBBPI, HD1, ATPb ATPb (ATP synthase CF1 beta chain). Comparative sequence analyses of measure purity and concentration of the genome regions containing these genes will facilitate understanding the extracted DNA. Figure 9: Represntation of mutiple tillers (A), what makes Oryza glaberrima so resilient to harsh conditions and how the Genes were identified and primers exmple of tissue sample (B) mixtures of O. sativa and O. glaberrima genomic backgrounds may be in- Future Directions were designed for these genes to volved. Identifying the genetic origins of the stress tolerance of Oryza Once plants grow to tissue harvest stage and provide enough material compare O. glaberrima to O. sativa. glaberrima will allows us to ap- for genomic DNA extractions, they will be whole genome sequenced The loci are associated with salt tol- plythese traits to other rice using long-read PacBio sequencing. These sequences will be analyzed erance (HTK1), drought tolerance crops to create a more sustain- to show the genetic relationship between O. glaberrima (African rice) (OsDREB2A), antiherbivore defense able crop in varying areas. and the rice of Trinidad plus possibly Carolina Gold rice. These analy- mechanism (OsBBPI), flowering ses will plot the origins of domestication backgrounds and the evolu- time (HD1), and ATP synthase CF1 tion of the Trinidad rice. This dataset will also include Carolina Gold beta chain (ATPb). PCR was per- rice because its origin is also still unknown but records suggest a small formed for these loci in O. sativa, O. portion of the genome may come from O. glaberrima and the vast ma- glaberrima and a negative control. jority of the genome derived from O. sativa. In addition to under- The sample was then run in a gel to standing the history of Trinidad rice and Carolina Gold rice, the stress look at the different lengths of the resilience of O. glaberrima can be applied to other types of plants, in- gene. The gel was run at 120 Volts in cluding Trinidad rice. Abiotic stress tolerances will be tested in Trini- a 1% 0.5x TBE solution for 40 min- utes. References: Figure 3: Map of Trinidad dad rice genotypes in future studies. With further understanding of Bender, William; "Population, Food, and Nutrition." 1997 the genome regions containing these loci, we plan to contribute to- Li, Zhi-Ming; "Genetic Diversity and Domestication History of African Rice (Oryza Glaberrima) as Inferred from Multiple Gene Sequences." 2011 Figure 4: Map of locations of Oryza glaberrima were provided wards developing rice varieties that provide locally adapted, sustain- Wang, Muhua; "The Genome Sequence of African Rice (Oryza Glaberrima) and Evidence for Inde- able cropping systems resilient to climate change pendent Domestication." 2014