Genetic Diversity and Cryopreservation of the Endangered Wild Rice Zizania texana

1. Genetic Diversity and Cryopreservation of the Endangered Wild Rice Zizania texana Christopher M. Richards1, Ann A. Reilley1, Christina Walters1, Darren Touchell1, Adam Henk1, and Michael F. Antolin2 1 USDA-ARS National Seed Storage Laboratory, Fort Collins, CO 2 Colorado State University, Fort Collins, CO The authors wish to acknowledge the support of the US Fish and Wildlife Service. Texas wild rice (Zizania texana) is a critically endangered plant species endemic to the San Marcos River in central Texas. Due to the accelerated pace of riparian habitat loss, the USFWS recovery plan for this species includes the establishment of an ex situ collection to stem the pace of genetic erosion. A total of 89 clones were removed from the various river populations and grown in a conservation collection in a former fish hatchery. Over the last 4 years, about half of the individuals in this collection have died. The difficulty in maintaining a living collection has increased the need to conserve the remaining genetic diversity of this species in seed banks. The seeds of Z. texana are recalcitrant and are therefore impossible to store using conventional protocols. Methodologies for their cryo-storage, worked out in our lab, give 50-70% survival following exposure to liquid nitrogen. The integration of both in situ and ex situ methods within a larger conservation and recovery effort depends in part in on minimizing the loss of natural genetic diversity associated with sampling bottlenecks. It is vital that the genetic diversity of the seed collections is not altered through the process of cryopreservation. To address this issue, we developed a set of highly variable SSR markers to evaluate shifts in the genotypic frequencies observed before and after cryopreservation. We present data that demonstrates the efficacy of cryopreservation in maintaining genetic diversity in ex situ collections. In addition, we show that these data represent the first step in larger evaluation of this species’ spatial and genetic structure and that many aspects of Z. texana’s biology lend themselves as a model system for studies of conservation genetics in other species. Objectives The current study focuses on the genetic component of cryo-preservation. Specifically, does cryo-preservation impose genetic selection in naturally diverse wild germplasm? Natural History Zizania texana is one of three species of wild rice currently recognized in North America. Unlike its more widespread congeners (Z palustris and Z. aquatica), Z. texana is now endemic to a 4km section of the upper San Marcos River and has been listed as an endangered species by the US Fish and Wildlife Service since 1985. Individuals grow in clonal mats in the shallow river-bed. Its isolation from other rice populations (900 km west and 1500 km south of the nearest known populations of wild rice) is somewhat a mystery. Although there are numerous spring-fed riparian habitats similar in quality to the San Marcos River along the Balcones fault in Central Texas, none support populations of Z. texana. It has been postulated that the current range represents a relict of a much wider distribution. Molecular phylogeny of the genus places Z.texana cluster close to Z. palustris (Northern wild rice) however, Z. texana shows many distinct differences in life history traits (perennial and clonal vs. annual) and seed dispersal traits (it lacks a head shattering trait typical of Northern wild rice). Material and Methods Seeds from flowering plants, maintained in a common garden at Southwest Texas State University, were randomly collected, pooled and split into two groups. The first group of seeds was extracted for DNA directly whereas the second group was put through the modified cryo-storage procedure before DNA was extracted. DNA samples from seeds (dissected embryos) were extracted using a Qiagen (Qiagen, Inc., Valencia, CA) anion exchange resin (DNeasy plant System) and served as template for PCR using primers specific to 2 micro-satellite loci. The resulting amplification products were resolved using a Licor 2400 automated sequencer on a 6.7% acrylamide gel. Specific allele assignments were accomplished using SAGA genotyping software. Genetic analysis of the genotypic data was run on SAS (SAS Institute, Cary, NC) and Genetic Data Analysis (GDA, Paul Lewis, University of Connecticut). Embryos of Zizania texana embryos excised from grains Germinating seeds of Zizania texana Seed samples ============================== Locus Sample Size Obs_Het ============================== L01 118 0.2712 L21 118 0.8475 Mean 118 0.5593 St. Dev 0.4075 ============================== Cryo-preserved Samples ============================= Locus Sample Size Obs_Het ============================= L01 112 0.2143 L21 112 0.6879 Mean 112 0.5411 St. Dev 0.2147 ============================= Zizania spp. produce seeds that do not survive desiccation. This so-called recalcitrant physiology prevents preservation of the seeds by conventional storage protocols that require extreme drying. Cryopreservation protocols were developed based on principles of optimizing water contents and rapid cooling (about 100oC/sec) into subcooled liquid nitrogen. These procedures resulted in > 90% survival for embryos of Z. palustris but very low survival for embryos of Z. texana (below right). To improve Z. texana survival, cryoprotective procedures commonly used in the animal industry were adapted: embryo cells were loaded with a protective sugar and briefly exposed to a cryoprotective solution containing glycerol and DMSO (below left). The additional cryoprotection resulted in 60 to 80% survival (below right). Current studies investigate the basis for different responses to liquid nitrogen between closely related species and the effect of mortality on the genetic structure of the population. Conclusions From this preliminary data, it is evident that both the allelic frequencies and average heterozygosity are maintained after cryo-storage. The pairwise Fst values among the before and after samples were 0.018 ( 0.023) indicating that the change in genetic variance was very small and could be due to the 30% mortality of the cry-preserved samples. Future Directions This study represents the merely the first step in integrating an ex situ collection into the larger recovery plan for this species. In many ways, Zizania texana is a useful model system to address the common issues involved ex situ collections and restoration biology. We are in the process of describing the ecological genetics of the whole species along the river system, including the genetic structure, effective population size and the extent of individual clones. Currently we have ~30 additional loci to add to the data set. These data will be critical in developing a sampling strategy that results in an ex situ collection that closely represents the diversity in the natural system.