title: Development of Microsatellites for Population Genetic Analyses of the Granulate Ambrosia Beetle (Coleoptera: Curculionidae).
abstract: Limited male dispersal and local mating in ambrosia beetles are expected to result in extreme inbreeding and highly structured populations. In this study, we developed microsatellite markers for the granulate ambrosia beetle, Xylosandrus crassiusculus (Motschulsky), for use in future studies into population and family structure of this invasive pest species. We employed de novo next-generation sequencing to generate whole genome shotgun sequences for the characterization of microsatellite loci. Approximately 6% of the 84,024 contigs generated from Hi-Seq Illumina 2x250bp sequencing contained microsatellites with at least four repeats of di-, tri-, tetra-, penta-, and hexamers. Primers were synthesized for 40 microsatellite loci with trimer repeat units. Twenty-four primer pairs yielded consistent PCR products of unique loci and were validated for population genetic application using three sample groups each containing 20 X. crassiusculus individuals from Mississippi. Thirteen loci were found to be polymorphic with up to five alleles per population. The two beetle sample groups from Pearl River County (Poplarville and McNeill) belonged genetically to the same population. The population from Lamar County (Purvis) was genetically distinct, separated by a moderate genetic distance (FST = 0.11) and five unique alleles (with >5% frequency). Consistent with the perceived mating structure (incest of females with flightless males), the populations showed homozygote excess at most loci, as indicated by the coefficients of inbreeding (FIT = 0.45 and FIS = 0.37) and high mean relatedness among individuals (r = 0.15).
authors.affiliation: Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 ( chusseneder@agcenter.lsu.edu ; JSPark@agcenter.lsu.edu ; jpark16@cbnu.ac.kr ).|Corresponding author, e-mail: chusseneder@agcenter.lsu.edu.
authors.forename: Claudia
authors.initials: C
authors.lastname: Husseneder
authors.affiliation: Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 ( chusseneder@agcenter.lsu.edu ; JSPark@agcenter.lsu.edu ; jpark16@cbnu.ac.kr ).|S1-5 204b, Chungbuk National University, 1Chungdae-ro, Seowon-gu, Chengju, Chungbuk, 28644, South Korea.
authors.forename: Jong-Seok
authors.initials: JS
authors.lastname: Park
authors.affiliation: USDA-ARS, Thad Cochran Southern Horticultural Lab, 810 Hwy 26 W, Poplarville, MS 39470 (chris.werle@ars.usda.gov; John.Adamczyk@ars.usda.gov).
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Werle
authors.affiliation: USDA-ARS, Thad Cochran Southern Horticultural Lab, 810 Hwy 26 W, Poplarville, MS 39470 (chris.werle@ars.usda.gov; John.Adamczyk@ars.usda.gov).
authors.forename: John J
authors.initials: JJ
authors.lastname: Adamczyk
mesh_terms: D000818:Animals; D005260:Female; D049810:Genetic Speciation; D014644:Genetic Variation; D007178:Inbreeding; D008297:Male; D018895:Microsatellite Repeats; D008922:Mississippi; D012098:Reproduction; D017422:Sequence Analysis, DNA; D048889:Weevils / Q000235:genetics* / Q000502:physiology
keywords:  genetic marker; PCR; genetic diversity; inbreeding; next-generation sequencing