Abstract
Here we report a mammal sexing procedure based on the detection of quantitative differences between females and males in the X-linked loci (quantitative sexing, Q-sexing). This novel technique was validated using samples from Siberian tigers (Panthera tigris altaica) whose sexes were known. The Q-sexing technique relies on the fact that amplifications proceeding exclusively from the two X chromosomes in a female mammal should reach the threshold cycle (CT) in a real-time quantitative real time polymerase chain reaction (qPCR) assay sooner than amplifications from the single male X chromosome. Nevertheless, given that the amplification efficiency may vary between samples, results have to be calibrated to a marker that does not vary in copy number between the sexes (for example, an autosomal-linked locus). For this purpose we used quantitative real time polymerase chain reaction (RT qPCR) assays to quantify the amount of three specific Siberian tiger microsatellite markers (X-/Y- and autosomal-linked loci) in individual samples in order to determine the sex of an animal. A difference of one CT between the X and the autosome-linked loci was detected in males, but no such difference was present in female samples. The Q-sexing technique unambiguously separates female from male Siberian tigers. The future of RT qPCR is bright as technology is becoming ever more rapid, cost-effective, easier to use and capable of processing higher throughputs. Thus, we expect that our novel technique for animal sexing will have a wide applicability, although further studies are still needed to adapt it to other animal species using specific primers. Key words: Polymerase chain reaction (PCR), quantitative real time polymerase chain reaction (qPCR), quantitative sexing, Siberian tiger.
Highlights
Animal molecular sexing techniques are usually based on polymerase chain reaction (PCR) amplification of deoxyribonucleic acid (DNA) sequences from the Y
Sexing techniques are based on differences in the Polymerase chain reaction (PCR) yielded fragment of the X-Y homologous gene amelogenin from the X and Y chromosome (Sullivan et al, 1993)
Sexing technique methods are usually based either on the presence or absence of PCR amplification products, on the differences in band lengths and/or the resulting banding patterns on gel, and/or on differences in sequences (‘qualitative sexing’), which enable researchers to distinguish between X and Y chromosomes or W and Z chromosomes [restriction fragment length polymorphisms (RFLPs, Sacchi et al, 2004), amplified fragment length polymorphism (AFLPs, Griffiths and Orr, 1999), single strand conformation polymorphism (SSCPs, Ramos et al, 2009), microsatellite alleles (Nesje and Roed, 2000) and oligonucleotide-microarrays: Wang et al, 2008; Kalz et al, 2006)]
Summary
Animal molecular sexing techniques are usually based on polymerase chain reaction (PCR) amplification of deoxyribonucleic acid (DNA) sequences from the Y (mammal) or W (bird) chromosome-linked loci (Aasen and Medrano 1990; Longmire et al, 1993; Ellegren, 1996; Putze et al, 2007), with concomitant amplification of X or W, respectively. Sexing technique methods are usually based either on the presence or absence of PCR amplification products, on the differences in band lengths and/or the resulting banding patterns on gel, and/or on differences in sequences (‘qualitative sexing’), which enable researchers to distinguish between X and Y chromosomes (mammals) or W and Z chromosomes (birds) [restriction fragment length polymorphisms (RFLPs, Sacchi et al, 2004), amplified fragment length polymorphism (AFLPs, Griffiths and Orr, 1999), single strand conformation polymorphism (SSCPs, Ramos et al, 2009), microsatellite alleles (Nesje and Roed, 2000) and oligonucleotide-microarrays: Wang et al, 2008; Kalz et al, 2006)]. As amplification efficiency can vary among samples, results should be calibrated to a marker that does not vary in copy number between the sexes (autosomal-linked loci)
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