Molecular Markers

Construction of a linkage map is often the first step to characterize genome of an organism. First genetic linkage map was constructed by Alfred Sturtevant, a student of Thomas Hunt Morgan, in the beginning of the 20th century while working with Drosophila melanogaster. Genetic maps are based on linkage of different markers on a chromosome and segregation of these markers in progeny of natural or experimental crosses. Sturtevant, Morgan and the other early geneticists used phenotypic markers, e.g., eye colour in Drosophila because segregation was easy to detect. Obvious drawback of these markers was their small number which resulted in large gaps in linkage maps corresponding to vast physical intervals on chromosomes. Advances in molecular biology and recombinant DNA technologies allowed to overcome this problem by complementing morphological linkage maps with molecular markers. Molecular marker is any variation in DNA molecule segregation of which can be detected in a cross. Molecular marker does not need to have a phenotype or to be a cause of a phenotype, but it still can be linked to a locus that confers phenotype of some sorts. Segregation of a molecular markers follows Mendelian rules and the meyotic recombination allows construction of molecular marker maps.

Variation at DNA level can take different forms - simple base pair changes (Single Nucleotide Polymorphisms or SNPs), small insertions and deletions (indels), variation in different repeat numbers (microsatellites, minisatellites, VNTR, etc.), as well as larger polymorphisms including insertion or deletion of whole genes and even larger chromosome regions.

Detection of various types of DNA polymorphisms forms the basis of molecular marker technologies. This web page describes only a few of the most commonly used molecular marker technologies in a limited amount of detail.
Restriction Fragment Length Polymorphisms (RFLPs, Botstein et al. 1980) can be caused by SNPs that change restriction enzyme sites, indels that change the size of restriction fragments or other types of polymorphisms that change the pattern of digestion of genomic DNA with restriction enzymes . The RFLP technology is based on digestion of genomic DNA from different individuals or plant varieties with restriction enzymes, separation of fragments in agarose gel and detection of fragments of interest using specific DNA probes. One of the first whole genome linkage maps of barley based on molecular markers (mainly RFLPs) was constructed in 1993 (Kleinhofs et al. 1993). Since then RFLP has been instrumental in mapping of the barley genome, gene cloning and assessment of genetic diversity.
Polymerase Chain Reaction (PCR, Saiki et al. 1985) allowed to amplify specific regions of genomic DNA and then detect polymorphisms in these amplicons using restriction enzymes. This molecular marker technology is called Cleaved Amplified Polymorphis Sequences (CAPS, Konieczny and Ausubel 1993) . Availability of barley EST unigene and genomic sequences allowed to develop more than 200 CAPS markers with known position on linkage map (Rostoks et al. 2005).
Microsatellite (MS) or Simple Sequence Repeat (SSR) polymorphisms are caused by variation in short (1 - 6 bp) repeat number. PCR is used to amplify region containing the polymorphic repeat and then the resulting PCR amplicons are electrophoretically resolved to distinguish different SSR alleles (Weber and May 1989; Litt and Luty 1989). Each SSR locus can have several different alleles (varying number of repeat unit) which is advantageous for assessment of genetic diversity and variety identification for crop plant species. SSR polymorphisms have been widely used to map the barley genome (Ramsay et al. 2000; Varshney et al. 2007).
Amplified Fragment Length Polymorphism (AFLP) technology combines fragmentation of genomic DNA using restriction enzymes with selective PCR amplification (Vos et al. 1995). AFLP allows simultaneous detection of multiple polymorphic loci in the genome. The method does not require prior knowledge of genomic sequences, therefore it has been widely used to assess genetic diversity in wild plant species and minor crop plants lacking extensive sequence data bases. Due to its high throughput AFLP allows rapid generation of linkage maps. It is also possible to achieve ultra high marker density on linkage maps, e.g., recently a potato linkage map was generated with over 10,000 AFLP loci (van Os et al. 2006).
Single Nucleotide Polymorphisms (SNPs) are the most common type of genetic variation in any species studied to date. There are more than ??? million SNPs in human SNP data base and the estimated number of human SNPs is ??? (reference). SNP are usually biallelic markers, because of the low probability of mutation occuring at the same site multiple times. SNPs can be used as molecular markers to generate linkage maps by studying their segregation patterns in progeny (Kruglyak 1997). While each SNP site is less informative than SSR locus because of its biallelic nature, the abundance of SNPs makes them very useful both for linkage and genetic diversity studies. Combinations of SNPs (SNP haplotypes) are usually used in studies of genetic diversity and, thus, genome-wide studies require genotyping very large number of SNPs which necessitated development of high throughput SNP genotyping technologies. Currently there is a wide range of genotyping platforms suited for different number of SNPs and samples (Syvanen 2005). SNPs are particularly useful for genome-wide association studies, because of the high throughput marker systems. While SNP studies are common in human genetics, the advances in sequencing technology have allowed large scale SNP discovery also in crop plant species, such as barley (Rostoks et al. 2005). A collection of over 5,000 SNPs and indels was identified by resequencing genomic PCR amplicons (SCRI SNP data base) and many more were identified in barley EST (Expressed Sequence Tag) assemblies (Timothy J. Close, unpublished information). The available SNP information was used to design 1524 SNP genotyping array based on Illumina GoldenGate technology ( This pilot OPA1 (Oligo Pooled Array) was used to genotype more than 90 European spring and winter barley varieties, which allowed to study genetic diversity and population structure in barley, as well as the extent of linkage disequilibrium (Rostoks et al. 2006). Preliminary results indicated that association mapping in barley could be a viable alternative to linkage mapping in biparental mapping populations allowing mapping resolution sufficient for plant breeders. Currently, two complementary projects in UK (AGOUEB) and USA (Barley CAP) are carrying out association mapping in several thousand European (including 95 Latvian varieties and breeding lines) and North American barley varieties using two new OPAs containing over 3000 SNPs and phenotypic data from existing and new field trials.