A Microsatellite Multiplex Assay for Profiling Pig DNA in Mosquito Bloodmeals

Genetic profiling has been used to link mosquito bloodmeals to the individual humans, but this analysis has not been done for other mammalian bloodmeals. In this study, we describe a microsatellite-based method for identifying individual pigs in mosquito bloodmeals based on their unique multilocus genotypes. Eleven tetranucleotide microsatellites and a sex-specific marker were selected based on Smith-Waterman DNA sequence alignment scores from the reference genome and primers were designed with features that reduce primer dimers, promote complete adenylation, and enable fluorescent labeling of amplicons. A multiplex polymerase chain reaction (PCR) assay was optimized and validated by analyzing DNA of individual pigs from several nuclear families and breeds before it was used to analyze genomic DNA of pig-derived mosquito bloodmeals from villages of Papua New Guinea. Population analysis of the nuclear families showed high expected and observed heterozygosity. The probability of observing two unrelated or sibling individuals sharing the same genotype at a single microsatellite locus or a combination of loci was vanishingly low. Samples had unique genotypes and gender was accurately predicted. Analysis of 129 pig bloodmeals identified 19 unique genotypes, which varied greatly in frequency in the mosquito bloodmeal samples. The high allelic diversity of the microsatellite loci and low probability of false attribution of identity show that this genotyping method reliably distinguishes distantly and closely related pigs and can be used to identify individual pigs from genotyped mosquito bloodmeals.

Pengembangan Set Multipleks Penanda DNA Mikrosatelit untuk Analisis Variasi Genetik Padi dan Kedelai

<p>Detection of multiplex microsatellite markers in a<br />single capillary array on a laser detection system is traditionally<br />conducted with specific primers that are labelled with<br />fluorescent dyes. An alternative method using fluorescent<br />labels that are appended to 5’ end of universal primer M13<br />instead of to the specific primers offers flexibility in<br />designning multiplex panels and a less expensive method.<br />Allele size range of microsatellite loci that can be grouped in<br />multiplex panels can be accurately estimated by pooling and<br />analyzing DNA samples from several genotypes simultaneously.<br />This paper describes the procedure in development<br />of microsatellite multiplex panels using M13 fluorescentlylabelled<br />and estimation of allele size range based on pooled<br />DNA strategies. Two multiplex panels of PCR amplification<br />products for rice consisting of 15 loci and three panels for<br />soybean consisting of 10 loci have been designed. The<br />panels have been applied to 50 accessions of rice and soybean<br />with fairly good results. Further characterization of<br />allele size range, however, is required prior to the application<br />of these panels to diverse genotypes. The procedure<br />described here should be applicable in the development of<br />multiplex panels of other species.</p>