Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCR-RFLP

Determination of the species from which traces of source material, such as blood stains on a leaf, originate can sometimes be a difficult task in forensic DNA analysis. For instance, insurance claims that involve car accidents with animals require authentication. Species identification is also essential in food quality control-procedures or for the detection and identification of animal material in food samples. Numerous analytical methods that rely on protein analysis have been developed for species identification, such as electrophoresis techniques [1, 2], immunoassays [3] and liquid chromatography [4]. However, proteins are heat labile and lose their biological activity. Furthermore, their presence and characteristics depend on special cell types. Thus, for species identification, DNA analysis would be preferred over protein analysis. The first genetic approach for determination of species identity was the dot-blot technique [5]. At present, polymerase chain reaction (PCR) is the technique of choice for species identification [6]. Some PCR approaches are RAPD-PCR (random amplified polymorphic DNA fingerprints) [7] and others are focused on RFLP analysis [8]. In this work we present a more sensitive method to detect DNA from degraded samples. Usually, the required specimen (hair, a part of skin or a piece of meat) contains degraded DNA and the PCR products must be cloned before sequencing [9]. Existing techniques consist of laborious and costly DNA sequencing procedures. We therefore used a less time-consuming PCR method to amplify a short mitochondrial (mt) DNA fragment. The PCR-RFLP allowed discrimination between different species even in cases in which the source material contained only degraded DNA. The mitochondrial cytochrome b gene has been used in phylogenetic as well as in forensic investigations [10, 11, 12] and has been shown in a variety of studies to be a very useful DNA-region for species determination [13, 14, 15, 16, 17, 18]

However, before applicable for routine analysis, species-specific diagnostic polymorphisms or mutations must be determined.

If these mutations affect restriction enzyme sites, a simple PCR-RFLP can be used as a "sceening tool" for detection.

One area in which the PCR-RFLP screening tool would be useful is in training hunting hounds. Before hounds are accepted and approved for hunting, they have to be evaluated by different tests. These tests include an examination of the dog's ability for winding and trailing. To examine these abilities normally a track is prepared consisting of minute amounts of blood from game animals, e. g., wild boar, spotted on a few leaves that are distributed in the forest or field. A trained hound should be capable of winding and trailing without any problems. However, in a case that came to our attention, hounds were unable to wind and it was assumed by the owners that the examiner had used blood from domestic animals. Hence, the question as to the source of the track (i.e., blood from domestic or game animals) was open.

To solve this problem we established genetic test (PCR-RFLP) that focused on the use of diagnostic polymorphisms in the mitochondrial cytochrome b gene: Our method can be used for the discrimination between the following mammal species: cattle (Bos taurus), sheep (Ovis aries), goat (Capra hircus), roe buck (Capreolus capreolus) and red deer (Cervus elaphus).

For the analysis, DNA was prepared from blood remains on different leaves. The DNA was amplified as described in Methods. Figure 1 shows RFLP results of five different species (Bos taurus, Ovis aries, Capra hircus, Capreolus capreolus and Cervus elaphus) and the pattern of bands from the blood sample of one of the dry leaves. As controls, DNA-samples from known species were used. Specific RFLP patterns that distinguish between 5 species are analyzed here. To estimate the exact size of fragments, DNA sequence information was used. C. hircus shows a single fragment of 182 bp, whereas with C. capreolus a fragment of 162 bp was obtained. The RFLP pattern of C. elaphus consisted of 2 fragments of 108 bp and 54 bp respectively. The blood sample on the leaf shows four fragments: 114 bp and 68 bp-fragments, which are characteristic for B. taurus, and a 105 bp and 75 bp fragment which can be assigned to O. aries. This result indicates that the blood sample on the leaf represents a mixture from two species (B. taurus and O. aries). The results were obtained from two independent DNA extractions and confirmed several times by independent PCRs, as well as by DNA sequencing. Amplifications were repeated and species identification was verified by diagnostic Dloop sequencing [19]. The Dloop DNA sequence of the PCR amplified fragment of the blood track was aligned with the mtDNA sequence of B. taurus and three nucleotide differences were observed.

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In addition to species determination using PCR-RFLP analysis, we tested for different DNA ratios of mixed samples, by which an assignment to a species is still possible. A valid assignment of a mixture of B. taurus and O. aries is still possible for a ratio of 59:1(Fig. 1).

The data clearly illustrate that it is possible to identify the species from unknown material using PCR-RFLP, provided that comparison to a known species is performed on the same gel.

Furthermore, the PCR-RFLP enables the observation of frequent contaminants such as cattle DNA in routine diagnostic labs. While direct sequencing of coamplified endogenous DNA would lead to multiple sequences, the PCR-RFLP is able to separate two signals. Sometimes, possible contaminants (such as human DNA) can lead to false results, but with the designed primer pair this problem was circumvented. For this reason we designed maximally discriminatory primers and the mismatches in each primer are sufficient to exclude human DNA under stringent PCR conditions. The primers were tested with different amounts of added DNA. This method can be used for analysis of mixed samples, since up to three species in different proportions can be determined. The RFLP test using other tissues, e.g., muscle, hair with roots, bones, and saliva, yielded reproducible results. Faeces have not been tested so far.

However, when minimizing target DNA, the bands tend to fade away on the agarose gel. The presence of a specific PCR-RFLP for the species analysed, a fragment length of less than 200 bp and the exclusion of contaminating sequences improved methods for existing species determination. The principle of RFLP is often used in food analysis [20, 21]. However, the new PCR-RFLP method is capable of analysing degraded DNA, especially in forensic cases. Furthermore, the PCR-RFLP utilizes only a small fraction of apomorphic sites.

The speed and the efficiency of current nucleotide technology, such as automatic sequencing, will permit the identification of additional taxa. However, the establishment of a PCR-RFLP test would likely need further extensive experimentation.

In summary, we were able to show that a simple PCR-RFLP is efficient in differentiating between B. taurus, O. aries, C. hircus, C. capreolus and C. elaphus. However, after further development, the tested mitochondrial nucleotide sequences may allow the forensic identification of other animal species.