**5.5 Mitochondrial DNA markers**

*Synthetic Biology - New Interdisciplinary Science*

Single nucleotide polymorphisms (SNPs) are used as an alternative to microsatellites in genetic diversity studies. Several technologies are available to detect the type of SNP markers [23]. As biallelic markers, SNPs have relatively low amounts of information, and to reach the information level of a standard panel of 30 microsatellite loci, larger amounts must be used. However, ever-evolving molecular technologies increase automation and reduce the cost of typing SNPs, which will likely allow, in the near future, the parallel analysis of a large number of markers at a reduced cost. In this perspective, large-scale projects are being implemented for several livestock species to identify millions of SNPs [24] and validate several thousands and identify haplotype in the genome. As with sequence information, SNPs allow for direct comparison and joint analysis of different experiments. SNPs are likely to be interesting markers for future use in genetic diversity studies because they can be easily used in the assessment of functional or neutral variation. However, the preliminary phase of SNP discovery or selection of SNPs from databases is critical. SNPs can be generated through various experimental protocols, such as sequencing, single-stranded coformational polymorphism (SSCP) or denaturing high-performance liquid chromatography (DHPLC) or in silico, aligning and comparing multiple sequences from the same region from public databases on genomes and sequential expression tags (ESTs). If the data were obtained randomly, the standard population genetic parameter estimators cannot be applied. A common example is when SNPs initially identified in a small sample (panel) of individuals are then typed into a larger sample of chromosomes. By preferably performing sampling of SNPs at intermediate frequencies, such a protocol will affect the distribution of allele frequencies with respect to the probable values for a random sample. SNPs present a modern tool in the context of genetic analyzes of the population; however, it is necessary to develop statistical methods that will take

into account each SNP operating method and their locations [25, 26].

AFLPs are dominant biallelic markers [27]. Variations on many loci can be arranged simultaneously to detect single nucleotide variations of unknown genomic regions, where a given mutation may often be present in undetermined functional genes. The disadvantage is that they show a dominant mode of inheritance, which reduces their power during genetic analyses of the population on intraracial diversity and consanguinity. However, AFLP profiles are highly informative in the

Restriction fragment length polymorphisms (RFLPs) are identified using restriction enzymes that cut DNA only at specific "restriction sites" (e.g., EcoRI cuts at the site defined by the palindrome GAATTC sequence). At present, the most common use of RFLPs is downstream PCR (PCR-RFLP) to detect alleles that differ in sequence at a given restriction site. A gene fragment is first amplified using PCR and then exposed to a specific restriction enzyme that cuts only one of the allelic forms. The digested amplicons are usually resolved by electrophoresis. Microsatellites or SSRs (simple sequence repeats) or STRs (short tandem repeats) consist of a few nucleotides—2–6 base pair DNA sequence—epeated several times in tandem (e.g., CACACACACACACACA). They are spread on a eukaryotic genome. Microsatellites are relatively small in size and, therefore, are easily

**5.3 Amplification of fragment length polymorphism (AFLP)**

evaluation of race relations [28–32] and related species [33].

**5.4 Restriction fragment length polymorphism (RFLP)**

**5.2 SNP**

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Mitochondrial DNA polymorphisms (mtDNA) have been widely used in analyzes of phylogenetic and genetic diversity. The haploid mtDNA transported by the mitochondria of the cellular cytoplasm has a maternal mode of inheritance (the animals inherit the mtDNA from their mothers and not from their fathers) and a high mutation rate; it does not recombine. These features allow biologists to reconstruct intra and interracial evolutionary relationships by evaluating mtDNA mutation patterns. mtDNA tags can also provide a quick way to detect hybridization between farmed species and subspecies [36]. Polymorphisms in the hypervariable region of the D-loop or the mtDNA control region have largely contributed to the identification of wild ascendants of domestic species and to the establishment of geographical models of genetic diversity.
