Development and characterization of ten microsatellite loci for the eastern spadefoot toad, Scaphiopus holbrookii

Abstract

We isolated and characterized 10 microsatellite loci from the eastern spadefoot toad, Scaphiopus holbrookii. Loci were screened in 24 individuals from two schools of tadpoles in a single isolated wetland in South Carolina, USA. The number of alleles per locus ranged from 4 to 12, observed heterozygosity ranged from 0.200 to 0.875, and the probability of identity values ranged from 0.043 to 0.298. These new loci provide tools for examining the landscape genetics of a species facing continued destruction of its breeding habitat (small isolated wetlands) as well as fragmentation of upland life zones.Keywords Scaphiopus Á Spadefoot toad Á Microsatellite Á PCR primers Á SSR Á STR The eastern spadefoot toad (Scaphiopus holbrookii), a member of the family Pelobatidae, is found throughout much of the eastern US (Palis 2005). Adults are secretive, spending most of their time in underground burrows in sandy or loamy soils (Carr 1940;Pearson 1955). These pond-breeding amphibians typically use fish-free ephemeral wetlands, to which hundreds of adults may migrate during heavy rains in almost any month of the year (Hansen 1958;Neill 1957). Older tadpoles aggregate in schools of thousands of individuals (of unknown kinship) that forage voraciously throughout the pond (Scott 2008).A long-term mark-recapture metapopulation study (Greenberg and Tanner 2005) found limited inter-pond dispersal, but concluded that genetic analyses would be needed to understand metapopulation connectivity.We extracted total DNA from one S. holbrookii using the DNeasy tissue kit protocol (Qiagen, Valencia, CA) according to the manufacturer's instructions. We followed the enrichment procedure of Glenn and Schable (2005), with some exceptions. Briefly, DNA was digested with restriction enzyme RsaI (New England Biolabs), ligated to double-stranded linkers, denatured and hybridized to biotinylated microsatellite oligonucleotide mixes (mix 2 captured on magnetic streptavidin beads (Dynal). Unhybridized DNA was washed away and remaining DNA was eluted from the beads, amplified in polymerase chain reactions (PCR) using the forward SimpleX-6 as a primer. There were two primary changes to the Glenn and Schable (2005) protocol. First, a different linker was used (SimpleX-6 Forward 5 0 -AAAAG CACGAGCGGAACT and SimpleX-6 Reverse 5 0 -pAGT TCCGCTCGTGC). Second, the enriched libraries were sequenced on a 454 using titanium chemistry following standard Roche 454 library protocols (454 Life Sciences, a Roche company, Branford CT). Sequences were subjected to a 3 0 quality trim where only one base in the last 25 bases of the sequence contains a quality score less than 20 or alternatively contains one ambiguous base. CAP3 [33] was then used to assemble sequences at 98% sequence identity using a minimal overlap of 75 bp. Along with singlets, contigs of two or three sequences were searched for the presence of microsatellite DNA loci using the program MSATCOMMANDER version 0.8.1 (Faircloth 2008) and

Development and characterization of 18 microsatellite loci for the Southern Leopard Frog, Rana sphenocephala

“…DNA was then serially enriched twice for microsatellites using three probe mixes following Glenn and Schable (2005), with the changes described in Lance et al (in press) and used the SimpleX-2 linker (Henningsen et al in press). All methods for sequencing, microsatellite identification, primer design, and primer screening are as described in Erickson et al (2010).…”