Restriction site associated DNA markers

Restriction site associated DNA (RAD) markers are a type of genetic marker which are useful for association mapping, QTL-mapping, population genetics, ecological genetics and evolution. The use of RAD markers for genetic mapping is often called RAD mapping. An important aspect of RAD markers and mapping is the process of isolating RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction site of a restriction enzyme throughout the genome. Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms mainly in form of single nucleotide polymorphisms (SNPs). Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers.

The use of the flanking DNA sequences around each restriction site is an important aspect of RAD tags. The density of RAD tags in a genome depends on the restriction enzyme used during the isolation process. There are other restriction site marker techniques, like RFLP or amplified fragment length polymorphism (AFLP), which use fragment length polymorphism caused by different restriction sites, for the distinction of genetic polymorphism. The use of the flanking DNA-sequences in RAD tag techniques is referred as reduced-representation method.

The initial procedure to isolate RAD tags involved digesting DNA with a particular restriction enzyme, ligating biotinylated adapters to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, and isolating the biotinylated fragments using streptavidin beads. This procedure was used initially to isolate RAD tags for microarray analysis. More recently, the RAD tag isolation procedure has been modified for use with high-throughput sequencing on the Illumina platform, which has the benefit of greatly reduced raw error rates and high throughput. The new procedure involves digesting DNA with a particular restriction enzyme (for example: SbfI, NsiI,…), ligating the first adapter, called P1, to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, preparing the sheared ends into blunt ends and ligating the second adapter (P2), and using PCR to specifically amplify fragments that contain both adapters. Importantly, the first adapter contains a short DNA sequence barcode, called MID (molecular identifier), which allows to pools different DNA samples with different barcodes and to track each sample when they are sequenced in the same reaction. The use of high-throughput sequencing to analyze RAD tags can be classified as Reduced-representation sequencing, which includes, among other things, RADSeq (RAD-Sequencing).

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