Microsatellite documentation of male-mediated outcrossing between inbred laboratory strains of the self-fertilizing mangrove killifish (Kryptolebias marmoratus

Abstract Primers for 36 microsatellite loci were developed and employed to characterize genetic stocks and detect possible outcrossing between highly inbred laboratory strainsof the self-fertilizing mangrove killifish, Kryptolebias marmoratus. From attempted crosses involving hermaphrodites from particular geographic strains and gonochoristic males from others, 2 among a total of 32 surveyed progenies (6.2%) displayed multilocus heterozygosity clearly indicative of interstrain gametic syngamy. One of these outcross hybrids was allowed to resume self-fertilization, and microsatellite assays of progeny showed that heterozygosity decreased by approximately 50% after one generation, as expected. Although populations of K. marmoratus consist mostly of synchronous hermaphrodites with efficient mechanisms of internal self-fertilization, these laboratory findings experimentally confirm that conspecific males can mediate occasional outcross events and that this process can release extensive genic heterozygosity. The mangrovekillifish,Kryptolebias(formerlyRivulus)marmoratus, is the only vertebrate species known to reproduce uniparentally by internal self-fertilization Earlier molecular techniques including multilocus DNA fingerprinting have revealed extensive genetic variation in some natural populations of K. marmoratus Materials and Methods Microsatellite Development To enrich for microsatellites in a genomic library for K. marmoratus, we employed a modified version of a hybridization The hybridization solution was mixed with magnetic streptavidin beads (Invitrogen, Carlsbad, CA), and hybridized fragments were captured on a magnetic block. Enriched DNA, recovered by precipitation, was polymerase chain reaction (PCR) amplified (25 ll total reaction volume) under the following conditions: 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 2.0 mM MgCl 2 , 25.0 lg/ml bovine serum albumin, 0.2 mM each dNTP, 0.5 lM SuperSNX24 forward primer, and 0.5 units Taq DNA Polymerase (Promega, Madison, WI). The PCR product was ligated into a PCR 2.1-TOPO vector and transformed into One Shot Top10 Chemically Competent Escherichia coli cells. Positive colonies were screened for b-galactosidase activity using materials in the TOPO TA cloning kit (Invitrogen; Carlsbad, CA). Insert sizes were verified from positive colonies by PCR amplification with the M13 forward (À20) and reverse (À27) primers (0.5 lM final concentration). Inserts !500 bp were purified using ExoSAP-IT (United States Biochemicals, Cleveland, OH) and sequenced with Big Dye chemistry (version 3.1, Applied Biosystems, Foster City, CA) using M13 forward or reverse primer on an ABI 3100 Genetic Analyzer equipped with 80-cm capillaries. Sequences were edited using Sequencher version 4.1.4 (Gene Codes Corporation, Ann Arbor, MI), and primers flanking microsatellite regions were developed using OligoAnalyzer 3.0 (Integrated DNA Technologies, Coralville, IA; http://www. idtdna.com/Scitools/Applications/Primerquest/). Primers were tested by amplifying genomic DNA from adult K. marmoratus specimens in laboratory strains initiated many generations ago from single hermaphroditic specimens from Florida, Belize, or Honduras. PCR conditions were optimized for each primer pair, and one primer from each pair was labeled at the 5#-end in either of 2 ways: directly with a 6-FAM or HEX fluorophore (Integrated DNA Technologies) or by attaching a reverse tag (5#-GGAAACAGCTATGACCATG-3#) for tailed PCR with an M13 primer labeled with a 6-FAM, HEX, or NED (Applied Biosystems) fluorophore. The 36 sequences from which primers were developed to amplify microsatellite loci were deposited to GenBank (accession numbers DQ335412-DQ335447). Laboratory Crosses Crossing experiments involved highly inbred laboratory lines each propagated generation after generation by a single selffertilizing hermaphrodite (herm) and each tracing back to a single wild herm originally collected at Utila Island, Honduras (strains Hon2 and Hon7), Belize (Bel50.91), Florida's Everglades National Park (ENP12), or Brevard County, Florida (CCHA). Each attempted cross involved placing an old (nearly senescent) herm with a secondary male in a small culture dish containing 13% saltwater solution DNA Extractions High molecular weight genomic DNA samples were isolated using a standard guanidinium isothiocyanate (GIT) chaotropic salt procedure Genotypic Assays Amplifications of microsatellite loci with fluorescently labeled PCR primers were performed in a 12.5-ll reaction volume containing 1 ll of purified genomic DNA, PCR Master Mix (Promega), and 0.5 lM of both forward and reverse primers. Tailed PCRs were performed in a 10-ll volume containing 1 ll of purified genomic DNA, 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl 2 , 25.0 lg/ml bovine serum albumin, 0.2 mM each dNTP, 0.25 lM M13-labeled primer, 0.25 lM locus-specific primer, 0.025 lM tailed locus-specific primer, and 0.4 units Taq DNA Polymerase (Promega). PCR products (1.5 ll) were mixed with 2.5 ll deionized formamide, 0.5 ll of either GeneScan-400HD [ROX] or GeneScan-500[ROX] internal lane standard, and 0.5 ll loading dye. Samples were denatured in a 95°C heating block for 3-5 min and chilled on ice before being loaded onto 5% acrylamide gels. Samples were electrophoresed on an ABI 377 DNA Sequencer at 3000 V for 2 h at 55°C. Alleles were sized using the software packages GeneScan version