**ABSTRACT NOT FOR CITATION WITHOUT AUTHOR PERMISSION. The title, authors, and abstract for this completion report are provided below.  For a copy of the full completion report, please contact the author via e-mail at scribne3@msu.edu.  Questions? Contact the GLFC via email at slrp@glfc.org or via telephone at 734-662-3209.**




Nicholas Sard1,2, Seth Smith1, Jared Homola1, Jeannette Kanefsky1, Gale Bravener3, Jean Adams4, Chris Holbrook5, Pete Hrodey6, Kevin Tallon3, Kim Scribner1,7


1Department of Fisheries and Wildlife, State University, 480 Wilson Rd., 13 Nat. Res. Bldg. East Lansing, MI 48824

2 Biology Department, SUNY Oswego, Oswego, NY 13126

3 Fisheries and Oceans Canada, 1219 Queen Street E. Sault Ste. Marie, Ontario P6A 6N5

4 Great Lakes Science Center, USGS,1451 Green Road, Ann Arbor, MI 48105

5 USGS, 11188 Ray Road, Millersburg, MI 49759-9481

6 USFWS, 3090 Wright Street, Marquette, MI 49855-9649

7  Department of Integrative Biology,  State University, 480 Wilson Rd., 13 Nat. Res. Bldg. East

     Lansing, MI 48824


December 2018



This Technical Assistance Program (TAP) project was designed to develop large numbers of polymorphic single nucleotide polymorphism (SNP) DNA loci and apply these genomic tools to conduct pedigree analyses for larval sea lamprey of different ages collected by agency larval assessment personnel from Great Lakes tributaries.  Sea lamprey abundance and wounding in several areas of the Great Lakes have been higher than target levels over the past decade.  Annual assessments of adult spawner abundance and stock-recruitment relationships are difficult to obtain using traditional fisheries methods, particularly in larger Great Lakes tributaries.  Additional applications of data were explored using a geographically more expansive collection of spawning adult samples from Great Lakes tributaries.  We used restriction site associated DNA sequencing (RADSeq) to discover genetic variation in a subset of individuals from Duffins Creek and the St.Clair River and designed RNA baits complementary to variable loci. We use these baits in combination with a RAD-capture genotyping method to efficiently genotype 3446 RAD loci (encompassing 11,970 SNPs), thereby affording biologists the opportunity to utilize SNP markers in sea lamprey monitoring and research. We characterize levels of diversity within and among populations (mean inter-population Fst range 0.001 to 0.020) and demonstrate population genetic applications.  The newly published germ-line sea lamprey genome allowed mapping of all SNP loci to putative chromosomes so SNP independence (lack of physical linkage) could be established.  Outlier loci (loci highly differentiated among sampled populations – range of inter-population Fst 0.205 to 0.374) were identified.  Genome annotation allowed for identification of putative functions of genes at or near each outlier SNP locus.  We conducted simulation analyses to demonstrate that accuracy of the markers when used in pedigree or parentage analyses when different numbers of loci, different numbers of larvae and different numbers of spawning adults contributed to offspring.  We show that accuracy in pedigree assignment was very high when 200 or 500 loci are used, even in situations of high spawner abundance (e.g., 1000 adults). We estimated the number of adults consistent with the full- and half-sibling larvae samples in each of 3 consecutive cohorts from Duffins Creek in the Lake Ontario basin (N=17, 53, and 25 adults for N=38, 146, and 30 larvae sampled from cohorts 1-3, respectively).  All larvae from each of 2 cohorts from the St. Clair River had different parents. We found ‘hot spots’ or areas of high numbers of related larvae in that were consistent for the 1, 2, and >3 year cohorts in Duffins Creek, likely indicating year-to-year consistency in use of a primary spawning site.  We also demonstrated the ability to characterize dispersion of larvae from the same full and half-sibling families. Estimates of mean (and variance) adult reproductive success was 4.5 (53.5), 5.5 (113.1) and 2.4 (4.0) for adults producing larvae sampled from cohorts of ages 1-3, respectively. Estimates of the effective number of breeding adults producing larvae for each cohort from Duffins Creek were low and varied consistently as a function of skew in adult reproductive success.  The rapid SNP screening protocol is cost effective. Based on observed library clonality rates, the proportion of sequence reads maintained after demultiplexing, and the proportion of reads mapping to target loci, we predict that reagent, consumable, and sequencing costs will be approximately $10 per sample but will vary depending on  the mean depth of sequencing coverage desired. Sequences of RNA baits used for targeted SNP genotyping and locus characteristics including chromosome location and levels of diversity within and among populations are available from co-authors Sard or Scribner.