Assessing the RNA integrity in dry seeds collected from diverse endangered species native to the USA

Hannah M Tetreault^1*Zoe Zingerman¹Lisa Hill¹Shaimaa Ibrahim¹Joyce Maschinski²Katherine D Heineman³Christina Walters¹

• ¹National Laboratory for Genetic Resource Preservation, Agricultural Research Service (USDA), Fort Collins, Colorado, United States
• ²Center for Plant Conservation, Escondido, United States
• ³San Diego Zoo Wildlife Alliance, Escondido, United States

Dry seeds do not show obvious signs of life and so testing for viability, health and life expectancy can be challenging. Usually testing seed quality involves adding water and measuring metabolic capacity or growth potential by vital staining or germination assays. Importantly, most laboratory seed tests are intended to assay immediate viability, while most genebanks need tests that predict seed performance in the distant future. All currently available assays require considerable a priori knowledge of germination conditions and seeds large enough to dissect. Germination conditions are often unknown for seeds produced from wild species and are an important criterion for seed testing. We aimed to test the feasibility of adapting a new seed quality assay that measures RNA integrity and appears promising for cultivated species, to seeds from wild species. Most of the 100 wild species we included are rare or endangered and in need of preservation through genebanking, thus tests with long-term predictive value are needed. To determine the feasibility of measuring RNA integrity in seeds from wild populations, we compared the quality of RNA extracted from seeds that were recently harvested to those of the same species that have been genebanked for 16 to 41 years. Using standardized methods (i.e., commercially available kits), we demonstrate reliable characterization of RNA quality across a diverse group of plants, despite variation in germination requirements, seed morphology or composition. RIN (RNA Integrity Number) values were usually high across all samples and variables, attesting to both the quality of newly collected material as well as its maintenance under genebanking conditions. This study conclusively demonstrates the feasibility of reliably extracting and characterizing RNA from dry seeds collected from wild populations, regardless of a variety of seed traits and morphologies. Relationships between RNA quality and seed age and viability require further exploration.