Development and characterization of 16 microsatellite markers for the Louisiana pine snake, Pituophis ruthveni, and two congeners of conservation concern

Abstract

We isolated and characterized 16 microsatellite loci from the Louisiana pine snake, Pituophis ruthveni. Loci were screened in 24 individuals from locations throughout its distribution in Louisiana and Texas. 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 ranged from 0.043 to 0.298. We examined cross-species amplification at these loci in P. catenifer (bullsnakes and gopher snakes) and P. melanoleucus (pine snakes). These new markers provide tools for examining the conservation genetics of this species complex. Louisiana pine snakes face numerous threats: population densities are extremely low and their natural habitat has been severely altered and fragmented. In southern Canada, P. catenifer is at the northern extreme of its range and limited by the availability of suitable over-wintering sites.

First successful artificial insemination of the endangered Louisiana pinesnake, Pituophis ruthveni

“…First analysis was performed by a commercial company, Rare Genetics Inc. using the Illumina MiSeq system (Illumina Inc.) to sequence four polymorphic loci previously established for Louisiana pinesnakes (Piru23, Piru25, Piru27, and Piru42; Kwiatkowski et al, 2010) and the resulting contig for each hatchling was compared to all possible sires and both dams. Second, genotyping was performed by the authors using fluorescent fragment analysis at nine microsatellite loci (Piru13, Piru15, Piru16, Piru23, Piru25, Piru27, Piru34, Piru42, Piru48; Kwiatkowski et al, 2010) and paternity analysis was conducted in Cervus 3.0.7 (Kalinowski et al, 2007) using unknown parent model where both parents were set as unknown and parental pairs were assigned among all seven dams and five potential sires bred that year. Simulations were used to estimate the 95% and 80% probability of identifying the successful pair based on the Delta between the LOD scores for the top two parental pairs.…”

Conservation genetics of the eastern yellow-bellied racer (Coluber constrictor flaviventris) and bullsnake (Pituophis catenifer sayi): River valleys are critical features for snakes at northern range limits

“…DNA was quantified using a spectrophotometer (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA). We genotyped all individuals (n = 177 racers, n = 103 bullsnakes) at 10 microsatellite loci that had been previously developed for each species (racers, [ 31 ]; bullsnakes, [ 32 ]). PCR was performed in 25 μl reactions containing 1X PCR Master Mix (Norgen Biotek, Mississauga, ON, Canada), 2 μM forward and reverse primer (forward primer labelled with fluorescent marker) and 10 ng of template DNA.…”

“…For racers, the thermal cycling was conducted as follows: 94°C for 5 min; 30 cycles of 94°C for 30 s, annealing temperature for 45 s, 72°C for 45 s; 8 cycles of 94°C for 30 s, 53°C for 45 s, 72°C for 45 s; and a final extension step of 30 min at 72°C. For bullsnakes, either standard thermal cycling parameters or a touchdown protocol was used depending on the locus (see [ 32 ]). Standard thermal cycling parameters were conducted as follows: 94°C for 5 min, followed by 40 cycles of 96°C for 30 s, annealing temperature for 30 s, and 72°C for 30 s. Touchdown cycling parameters consisted of 95°C for 5 min, 20 cycles of 96°C for 30 s, annealing temperature of 65°C (decreasing 0.5°C per cycle to 55°C) for 30 s, 72°C for 30 s, and 20 cycles of 96°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Both positive and negative controls were run for all loci; template DNA from a well-characterized individual was used as a positive control.…”