Main methods for moleculr marker discovery

The fundamental methods of determination of molecular markers:

RFLP – restriction fragment length polymorphism. RFLP markers are received having fragmented DNA into restrictases. Polymorphism is observed in cases when the mutation of one base pair causes loss of the restriction location or an occurrence of an additional one, if due to insertion or deletion the size of the restriction fragment changes, as well as due to unequal crossing-over.

RAPD – random amplified polymorphic DNA; RAPD uses only one primer from random nucleotide sequence of 8 to 50 bp length, which also serves as direct and inverted.

SCAR – Sequence Characterized Amplified Regions. Continuation of RAPD analysis - SCAR elements, i.e. ends of characteristic RAPD fragments of 16 - 24 bp length.

SSR – simple sequence repeats (microsatellites). SSRs are polymorphic due to different number of recurrences.

ISSR – inter-simple sequence repeats. ISSR method is RAPD modification, when instead of random sequence primer a primer of microsatellite sequence is used.

AFLP – amplified fragment length polymorphism. AFLP is a system of DNA markers, allowing identifying the differences of nucleotide sequences according to the size of DNA fragments, fragmented for restrictases, selected according to characteristic markers and amplified applying PCR method.

EST – expressed sequence tags. mRNA is produced from certain parts of the plant, depending on the response of the plant into biotic or abiotic stress, according to which a more stable cDNA is synthesized. This DNA reflects genes which are characteristic to the plant at that time.

CAPS - Cleaved Amplified Polymorphic Sequence. It can be the continuation of RAPD analysis, using certain RAPD fragments for the production of primers identifying them and a more precise investigation. Amplified fragments are fragmented for restrictases.