Identification of robust microsatellite markers for wild pig fecal DNA

Abstract

Collection of fecal samples for use in a genetic capture-mark-recapture framework has become popular as a noninvasive method of monitoring wildlife populations. A major caveat to this process, however, is that fecal samples often yield low quality DNA that is prone to genotyping errors, potentially leading to biases in population parameter estimation. Therefore, considerable care is required to identify robust genetic markers, especially in hot or humid conditions that may accelerate DNA degradation. We identified microsatellite loci in wild pig (Sus scrofa) fecal samples that were robust and informative within warm, humid ecosystems. To examine how degradation affected genotyping success, we sampled pig feces across 5 days and calculated how the number of quantitative polymerase chain reaction (qPCR) cycles required to reach the fluorescent threshold (C t ) changed over time. We identified 17 microsatellite loci that had high polymorphism and amplification success and low genotyping error rates (0-0.050 per locus). In the degradation experiment, C t increased over the 5 days, but in the absence of rain, the majority of samples produced accurate genotypes after 5 days (2,211/2,550 genotypes). Based on the high amplification success and low error rates, even after 5 days of exposure to warm, humid conditions, these loci are useful for estimating population parameters in pig fecal samples. Ó

Assessing the utility of metabarcoding for diet analyses of the omnivorous wild pig (Sus scrofa)

“…We collected fecal material as either fresh scat collected from transects within 24 hr of defecation following the methodology of Kierepka et al. () (California; 19 individuals) or from fecal material taken directly from the colon of culled individuals (Texas and Florida; 14 and 15 individuals, respectively). California samples were collected from 31 July 2014 through 3 September 2014 and immediately placed on ice in the field, then frozen.…”

A comparison of cost and quality of three methods for estimating density for wild pig (Sus scrofa)

“…Elevation of the SRS ranges from 30-115 m above sea level. Climatic conditions of the SRS are described in Kierepka et al 71 and are generally characterized by warm temperatures and high humidity. Wild pigs have been lethally controlled at the SRS since 1952 in an effort to mitigate damage 72 .…”

“…We collected fecal samples along transects every other day for 12 days for a total of 6 sampling occasions per study site. Research by Kierepka et al 71 demonstrated that accurate genotypes could be attained from wild pig fecal samples with five days of environmental exposure, excluding rain; therefore, our sampling scheme should be minimally affected by exposure issues. As in Ebert et al 62 , we employed a modified adaptive cluster sampling protocol, which is a method designed to help detection of rare but clustered samples, described in 73 to maximize collection of fecal samples and account for social behavior of wild pigs.…”

“…Laboratory methods. Full details of laboratory methods can be found in Kierepka et al 71 . To conduct capture-recapture (CR) analyses on the genetic samples, we had to first produce microsatellite genotypes for individual identification from each fecal sample.…”

Genetic tools in the management of invasive mammals: recent trends and future perspectives

“…qPCR has the additional benefit of providing quality control to select optimal DNA samples for further analysis, such as sequencing and genotyping, thus allowing researchers to avoid wasting resources on poor‐quality samples that are unlikely to yield results. Kierepka et al () used qPCR to screen feral pig Sus scrofa faecal‐derived DNA prior to genotyping, to generate a robust capture–mark–recapture protocol in order to facilitate accurate estimates of abundance.…”