Creating genomic resources for environmental DNA assay development for threatened and endangered turtle species

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Creating genomic resources for environmental DNA assay development for threatened and endangered turtle species
Lillian Pavord
Dr. Nicholas Sard
Dr. Peter Rosenbaum
Lillian Pavord
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Lillian Pavord


Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Lillian Pavord.

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Creating genomic resources for environmental DNA assay development for threatened and endangered turtle species Lillian Pavord, Dr. Peter Rosenbaum, and Dr. Nick Sard Biological Sciences Department, SUNY Oswego, Oswego, New York Background Habitat destruction and fragmentation has led to the drastic decrease in freshwater turtle populations. 1 The Bog Turtle ( Glyptemys muhlenbergii ), Wood Turtle ( Glyptemys insculpta ), and Spotted Turtle ( Clemmys guttata ) are critically endangered. 2,3,4 These species are difficult to monitor and manage because they are evasive and live in small and isolated populations. Genetic techniques, such as environmental DNA (eDNA) assays, are being used to survey populations. eDNA assays are used to make accurate estimates in distribution and relative abundance. 5 eDNA assays would greatly contribute to conservation efforts associated with freshwater turtle species. The three endangered species (above) lack fully sequenced mitochondrial genomes (mitomes ), which are needed for eDNA assay development. Dr. Rosenbaum has a large collection of turtle blood samples S amples comprise eight different turtle species, including the endangered species (above). Extracted DNAs will used in the development of eDNA assays that could help with freshwater turtle conservation efforts. Objectives Sequence the extracted turtle DNAs and assemble the mitomes of five turtle species. Use mitomes as a resource for eDNA assay development for the future surveys to detect and estimate relative abundance of threatened and endangered turtles. General methods Compile samples into a database so individuals can be identified. Extract DNA from turtle blood of individuals using a DNEasy Qiagen kit for blood and tissue samples. Prepare extract DNAs for sequencing using NEBNext Illumina kit. Have prepared DNA sequenced using an Illumina HighSeq 4000 offsite. Use results to assemble mitomes using assemblers optimized for bacterial genomes e.g., SPAdes. 6 Planned research 1) Several turtle species sampled across New York State Locations are shown ambiguously to ensure limited harm for the threatened and endangered species. Discussion and Future work A total of 80 Bog Turtles have extracted DNAs. Future work will extract DNAs from individuals from other species. High quality DNAs will be selected as individuals to be prepared and sent for sequencing at Novogene. Mean DNA concentration thus far is 64 30 ng/µl (1 standard deviation). Maximum and minimum values ranged from 120 to 6 ng/µl. Results suggest that most samples have high DNA concentrations. Mean A260/A280 ratio was 2.0 0.1, suggesting that DNAs were generally of high quality. Given that the year blood samples were collected varied over two decades, we sought to test if DNA concentration was correlated with preservation period. Results suggest a weak correlation (R 2 = 0.28). Once libraries are prepared and sequenced, we will assemble the mitomes. Mitomes assembled for species with already published mitomes can be used to compare to those we assemble to assess our de novo assembly approach. Mitomes will be used as a genetic resource to develop eDNA assays to detect a range of turtle species. References 1. Snell, E. A. (1987). Wetland distribution and conversion in southern Ontario. Inland Waters and Lands Directorate , 48 . 2. Stanford, C. B. (2018). Turtles in Trouble: The World's 25+ Most Endangered Tortoises and Freshwater Turtles 2018 . Turtle Conservation Coalition. 3. Brown, D. J., Cochrane, M. M., & Moen, R. A. (2017). Survey and analysis design for wood turtle population monitoring. The Journal of Wildlife Management, 81 (5), 868 877. 4. Howell, J. H., McKnight, D. T., & Seigel, R. A. (2016). A novel method of collecting spotted turtles ( Clemmys guttata ). Herpetological Review, 47 (2), 202 205. 5. Schwartz, M. K., Luikart , G., & Waples , R. S. (2007). Genetic monitoring as a promising tool for conservation and management. Trends in ecology & evolution, 22 (1), 25 33. 6. Bankevich , A., Nurk , S., Antipov , D., Gurevich , A.A., Dvorkin , M., Kulikov, A.S., Lesin , V.M., Nikolenko , S.I., Pham, S., Prjibelski , A.D. and Pyshkin , A.V. (2012). SPAdes : a new genome assembly algorithm and its applications to single cell sequencing . Journal of computational biology, 19 (5), 455 477. Acknowledgements Special thanks to Peter Rosenbaum for his work over the past three decades to develop this collection of samples and allowing me the chance to work with them. Also thanks to Scholarly and Creative Activity Program for funding the extraction of the Bog Turtle species to help their conservation effort. 2) Full mitome sequences are lacking have their mitomes sequenced. 3) Schematic of proposed approach The steps leading to the sequencing process and intended use of sequenced mitomes. Figure 1. Map of New York State with marked counties representing regions where turtles have been sampled. Table 1. List of the common and scientific names of the eight species in Dr. samples, as well as how many complete mitomes are published for each species on GenBank and how many sequences were published at the 12S, 16S, and COI loci. Learn more at Corresponding author: n Catalogue blood samples and identify individuals Extract DNA from blood using a Qiagen kit Quantify DNA extracted using a Nanodrop 2000 Select good quality extractions from 7 different species to be sequenced Prepare selected extractions for sequencing using Illumina Prep kit Send prepared extractions to an offsite Illumina sequencing machine Receive sequencing results and assemble mitomes for each individual sample used Publish assembled mitomes to be used as a resources for eDNA assays Use sequenced mitomes to enhance eDNA assay protocols Common name Scientific name Mitomes 12S 16S COI Emydoidea blandingii 0 0 0 8 Bog Turtle Glyptemys muhlenbergii 0 0 0 4 Box Turtle Terrapene carolina 0 1 3 29 Musk Turtle Sternotherus odoratus 0 3 5 12 Painted Turtle Chrysemys picta 4 1 4 12 Snapping Turtle Chelydra serpentina 3 9 5 13 Spotted Turtle Clemmys guttata 0 0 0 7 Wood Turtle Glyptemys insculpta 0 1 3 4