Printing: Microsatellite Primer Design and Testing for pebblesnails Daniel McCullough and Hsiu Ping Liu | Metropolitan State University of Denver, Department of Biology | Questions: dmccull5@msud enver.edu Objective Figure 2: Microsatellite sequence of loci Flusp_104214. Microsatellite sequence is indicated by the yellow highlighted nucleotides. Primers are the green highlighted nucleotides . The purpose of this research is to design, test and optimize microsatellite primers for pebblesnails. These primers can be used to study population structure and manage these relatively unknown species for biodiversity and conservation purposes. Fluminicola is a genus of freshwater snails in the Family Fluminicolidae found in western United States, they are commonly known as the pebblesnails ( Figure 1 ). Currently, there are 25 recognized species found in the Pacific Coast Watershed, from northern California to southern British Columbia. Several petitions have been filed to enter pebblesnails on the endangered species list. However many populations have never been formally described and most species have barely been studied ( Hershler et al., 2017). This project could not have been completed without the tremendous moral and funding support from Center for Advanced Stem Education and CO WY AMP. I would also like to thank the Biology Department for providing the lab space necessary to perform the research. Retest difficult to score dinucleotide loci by Determine the variability and utility in other pebblesnail species. DNA Extraction Step 1 Primer Design Step 2 Polymerase Chain Reaction (PCR) Step 3 M13 Labeled Fragment Analysis Step 4 Microsatellites are made up of short tandem repeats ranging in length from 2 6 base pairs. It is typically repeated 5 50 times. The relatively large number of alleles make microsatellite markers useful for conservation and population genetics studies. Ninety one microsatellites loci were tested and 42 loci (46.2%) were successfully amplified with expected fragment size. Forty two forward primers were tailed with M13 sequences, including 9 dinucleotide, 11 trinucleotide, and 22 tetranucleotide loci. Among the 18 loci tested using genetic analyzer, 12 loci can be scored easily (66.7%) ( Figure 6a, 6b ). Five loci were tetranucleotide repeats and 4 were trinucleotide repeats, the last 3 were dinucleotide repeats. Microsatellites : Our results indicated that tetranucleotide and trinucleotide loci have a higher percentage of amplification. Nine out of 29 dinucleotide (31%), 11 out of 30 trinucleotide (36.7%) and 22 out of 32 tetranucleotide (68.8%) loci can be amplified. Fragment analysis: Among 18 tested loci, the dinucleotides were more difficult to score. Dinucleotide repeat means each repeat has two base pairs. The plus A phenomenon can make dinucleotide loci difficult to score. Ballard et. al., (2002) suggested that adding GCTTCT at 5 prime end of reverse primer can alleviate the scoring issue. M13 fluorescent labeling method : M13 labeling is cost effective way to perform fragment analysis. To order 24 fluorescent primers would cost $2400 ($100 per primer*24 primers=$2400),however using the M13 method it only costs $272 (40 base pairs*$0.18*24 primers=$172.80+1 fluorescent labeled primer). DNA was extracted from 3 species of pebblesnails Fluminicola modoci, Fluminicola multifarius, and Fluminicola klamathensis using CTAB method (Bucklin 1992). Primer design: primers are 18 24 base pairs long. The primers were designed based on the desired DNA fragment size, appropriate GC percent, and avoiding hairpin structure ( Figure 2 ). Primers were tested and optimized using polymerase chain reaction (PCR) with temperature gradients ( Figure 3 ). 1.5% agarose gel was used to visualize the PCR products ( Figure 4 ). For successfully amplified microsatellite loci, forward primers were tailed with M13 sequence (CACGACGTTGTAAAACGAC). PCRs were performed with M13 primer labeled with fluorescent molecule, M13 tailed forward primer, and reverse primer to generate fluorescent labeled DNA fragment ( Figure 5 ) (Boutin Ganche et at 2001). Fluorescent PCR products were run on the genetic analyzer ABI 3500 and analyzed using Gene Mapper. Ballard, Linda & Adams, Pamela & Bao, Yongde & Bartley, Duane & Bintzler , Doug & Kasch , Laura & Petukhova , Lynn & Rosato , Caprice. (2002). Strategies for genotyping: Effectiveness of tailing primers to increase accuracy in short tandem repeat determinations. Journal of biomolecular techniques : JBT. 13. 20 9. Boutin Ganache, I., Raposo , M., Raymond, M., and Deschepper , C.F. (2001) M13 tailed primers improve the readability and usability of microsatellite analyses performed with two different allele sizing methods. Biotechniques 31: 24 28. Bucklin, A. Use of formalin preserved samples for molecular analysis. Newsletter of Crustacean Molecular Techniques 2: 3. Hershler , R., Liu, H P., and Hubbart , N. (2017) Two new species of Fluminicola ( Caenogastropoda , Lithoglyphidae ) from southwest Oregon, USA, and a range extension for F. multifarius . ZooKeys 679: 1 20. Polymerase Chain Reaction (PCR) Fact Sheet. Genome.gov , www.genome.gov /about genomics/fact sheets/Polymerase Chain Reaction Fact Sheet. Schuelke , Markus. (2000). An economic method for the fluorescent labeling of PCR fragments. Nature biotechnology. 18. 233 4. 10.1038/72708. Zane, L., Bargelloni , L. & Patamello , T. 2002. Strategies for microsatellite isolation: a review. Molecular Ecology 11(1): 1 16. Figure 5: Depicts how fluorescent labeled DNA fragments are created. ( Schulelke , 2000). Figure 3: Depiction of how polymerase chain reaction works. Figure 4: Flusp 104214 on 1.5% gel electrophoresis showing DNA products from gradient analysis. Figure 6. Chromatogram for loci Flusp_33873 and Flusp_72029. 6a shows a heterozygous individual in the pebblesnail species F. modoci . 6b shows a homozygous individual in pebblesnail species F. multifarious. 6c demonstrate a difficult to score allele. Since microsatellite primers are typically species specific and none are available for the pebblesnails , new primers must be developed for each species, this is a time consuming and extremely costly steps of all such studies (Zane et al., 2002). The goal of this project is to design, test, and optimize microsatellite primers for pebblesnails . Figure 6a Figure 6b Figure 6c Results Works Cited Acknowledgements Methods and Materials Introduction Future Work Discussion Figure 1: Pebblesnails preserved in 70% ethanol.