healthmedicinet

The brown tree snake Boiga irregularis was inadvertently introduced to the island of Guam following World War II 1] and has devastated native avifauna. There is presently only one study examining the
genetic composition of source populations using DNA sequence data from mitochondrial
and nuclear sequence loci 2]. Therefore, highly polymorphic multilocus markers are needed to investigate the genetic
ramifications of invasive species demography and population genetics of B. irregularis.

To investigate the potential use of population genetics tools to inform invasive species
management by investigating source populations or determining the current population
structure of B. irregularis on Guam, we developed microsatellite markers for B. irregularis. Genomic DNA used to isolate the microsatellite loci was extracted from one individual,
utilizing a QIAamp DNA microkit (QIAGEN). An Illumina paired-end shotgun library was
prepared by following the standard protocol of the Illumina nextera DNA sample preparation
kit and using a dual index identifier adaptor. Illumina sequencing was conducted on
the HiSeq with 100 bp (base pair) paired-end reads. The DNA libraries were prepared
to range from 300–600 bp and then paired end reads were performed on the resulting
fragments. Thus we sequenced 100 bp from end to end, with primers designed on the
reads (one from each) as a majority of the time the two sequences did not overlap,
leaving an unknown length of unsequenced bases between the reads. Using the program
PAL_FINDER_v0.02.03 3], 190,804 reads containing tetra, penta, and hexanucleotide repeats were identified
and batched to a local installation of the program Primer3 (version 2.0.0) for primer
design.

Forty-eight primer pairs were tested for amplification and polymorphism following
the methods detailed in O’Bryhim et al. 4]. Amplification reactions in 12.5 µl contained 20 ng of genomic DNA, 10 mM Tris pH
8.4, 50 mM KCl, 25.0 µg/ml BSA, 0.4 µM unlabeled primer, 0.04 µM CAG-tag labeled primer,
0.36 µM universal dye-labeled primer, 3.0 mM MgCl
₂
, 0.8 mM dNTPs, using an applied biosystems GeneAmp 9700. The forward primer from
each locus was 5? modified with an engineered “CAG-tag” sequence to enable use of
a third, fluorescently labeled primer in PCR following Schuelke 2000 5]. Touchdown thermal cycling programs encompassing a 10 °C span of annealing temperatures
ranging between 65 and 55 °C were used for all loci. Touchdown parameters consisted
of an initial denaturation step of 5 min at 95  C followed by 20 cycles of 95 °C for
30 s, highest annealing temperature (decreased 0.5 °C per cycle) for 30 s, and 72 °C
for 30 s; and 20 cycles of 95 °C for 30 s, lowest annealing temperature for 30 s,
and 72 °C for 30 s, and a final extension at 72 °C for 5 min. PCR products were run
on an ABI-3130xl sequencer and sized with Naurox size standard prepared as described
in DeWoody et al. 6], except that the unlabeled primer for the Naurox standard was GTTT pig-tailed. Results
were analyzed using GeneMapper version 3.7 (Applied Biosystems). Thirty-three of the
tested 48 primer pairs amplified high quality PCR product that exhibited polymorphism
and consistent allele base pair amplification.

We assessed the variability of the 33 polymorphic loci for 32 individuals from Guam.
Conditions and characteristics of the loci are provided in Addditional file 1: Table S1. Cervus version 3.0.6 was used to calculate the number of alleles per locus
(A) and observed and expected heterozygosity (H
_o
and H
_e
) 7]. All 33 loci were determined to be polymorphic, with a range of 3–10 alleles per
locus with an average of 4.62 (Additional file 1: Table S1). Probability of identity (PI) was estimated using GenAlEx v6.5 8]. Deviations from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were
performed using GENEPOP v4.0 9]. After Bonferroni correction for multiple comparisons, eight loci showed significant
deviations from HWE (? = 0.00139), and linkage disequilibrium was detected for 1 of
the 630 paired loci (? = 0.0000794). Probability of identity values ranged from 0.043
to 0.539. These new microsatellite loci are ideally suited to address important questions
including testing the efficacy of management and control techniques for B. irregularis on Guam by providing markers ideal for assessment of population structure before
and after eradication efforts.