marker-assisted selection in wheat

34Marker-assisted selection – Current status and future perspectives in crops, livestock, forestry and fishto multiplex (combine) several markerswith non-overlapping size ranges on asingle electrophoresis run. The results arehighly reproducible, and the markers areeasily shared among researchers simply bydistributing primer sequences. AlthoughSSRs are abundant in most eukaryoticgenomes, their genomic distribution mayvary. Uneven distributions of microsatelliteslimit their usefulness in some species.Inter-SSRs (ISSRs) are another type ofmolecular marker that makes use of microsatellitesequences. ISSRs use PCR primersanchored in the termini of the repeatsextending into the flanking sequence by severalnucleotides (Zietkietkiewicz, Rafalskiand Labuda, 1994). PCR products are producedfor each pair of microsatellites thatare in sufficient proximity for PCR tooccur, or may be generated by anchoringone primer in the SSR motif and using asecond “universal” primer correspondingto a sequence that has been ligated ontothe ends of restriction fragments (as in theAFLP technique described above, wheregenomic DNA is first digested with arestriction enzyme and oligonucleotideadaptors are ligated to the ends of eachrestriction fragment, except that one primerresides in an SSR motif that is bracketedby the restriction sites) (Gupta et al.,1994; Goodwin, Aitken and Smith, 1997).Markers at multiple loci are assayed as thepresence or absence of bands of particularsizes. ISSRs can be visualized on agarosegels, on silver stained polyacrylamide gelsor fluorescently labelled for detection withan automated DNA sequencer.Transposable element-based markersTransposable elements (TEs) are anotherrapidly changing feature of the genomethat can be exploited as a source of variabilityfor molecular markers. Discovery ofTE sequences is a prerequisite for their useas markers. While TEs may be discoveredas mutations in alleles of genes conferringmutant phenotypes, they have also beendiscovered directly in genomic sequence(reviewed by Feschotte, Jiang and Wessler,2002). Transposon display is a modifiedAFLP procedure that differs only in thatone of the two primers is designed withinthe consensus sequence of a TE family sothat amplification depends on the presenceof a TE insertion within a restrictionfragment (Casa et al., 2000). Using thisapproach, the presence or absence of a TEcan be assayed simultaneously at manyloci throughout the genome. To assay fora TE insertion at a specific locus, singlecopy “anchor markers” can be designedwith primers located in unique sequencesflanking the region of interest. A size polymorphismindicates the presence or absenceof the TE in that particular location. Anchormarkers are advantageous because they areco-dominant, can be run on a simple agarosegel system and are biologically informativein that they provide evidence of bothcomplete, or incomplete, insertion or excisionevents. This methodology can also beapplied to any known indel feature regardlessof whether or not it is derived from aTE.Single nucleotide polymorphismsSingle nucleotide polymorphisms (SNPs)are an abundant source of sequence variantsthat can be targeted for molecularmarker development. Of all the molecularmarker technologies available today,SNPs provide the greatest marker density.SNPs are often the only option forfinding markers very near or within agene of interest, and can even be used todetect a known functional nucleotide polymorphism(FNP). Discovery of SNPs