DNA sequence information is providing a growing resource for the identification of genetic markers for use in plants. The establishment of ITEC has made significant numbers of ESTs available for wheat and barley. EST databases have provided an important source for SSR or microsatellite markers from a range of plant species. SSR markers derived from ESTs may have advantages over those from traditional enriched genomic libraries. Wider transferability of these SSRs may be possible because of higher sequence conservation within ESTs. Mapping of the SSR provides a map location, in many cases, for a gene of known function. Single nucleotide polymorphism markers can also be designed from EST information. This provides a rich source of DNA polymorphism for use in genome mapping. We have designed molecular beacons to detect SNPs in barley. The relative value and potential application of SSR and SNP markers in barley will be discussed.
Background: Microsatellite markers are valuable in genotyping and evaluation of genetic resources (WU and TANKSLEY 1993; SAGHAI MAROOF et al. 1994; GARLAND et al. 1999: MAGUIRE et al. 1999). ITMI initially focused on the mapping of genomes in the Triticeae using RFLP markers. More recently microsatellite or SSR markers have been more widely used because of their ease of analysis. These microsatellites have mostly been isolated from enriched genomic libraries. This process is laborious and expensive. The growing availability of DNA sequence information provides new opportunities for the development of molecular markers for use in the Triticeae. The recent completion of the sequence of the rice genome provides an important source of data. The International Triticeae EST Consortium (ITEC) provides a new source of species-specific sequence information from which markers can be derived. We have investigated the identification of microsatellite and single nucleotide polymorphism (SNP) markers in EST sequences. Two main options for the development of microsatellite markers are to source them directly from the target species or to identify them based upon information from a closely related species. Research at the Centre for Plant Conservation Genetics at Southern Cross University has addressed the isolation and characterisation of microsatellite loci from many plant species. Large numbers of loci have been identified in some species by producing enriched genomic libraries (ROSSETTO et al. 1999). In some cases such as sugarcane this has required optimisation of the microsatellite enrichment technique (CORDEIRO et al. 1999). Other species, such as pine trees have required screening of enriched libraries to allow efficient recovery of microsatellite loci (SCOTT et al. 2000a). Microsatellites derived from genomic libraries have been difficult to transfer between species in many cases, although exceptions have been found in some plant groups. For example, microsatellites appear to be widely transferable within the Myrtaceae, possibly due to limited sequence divergence in this family (ROSSETTO et al. 2000). Microsatellite transfer between Pinus species has also proven to be a useful option. Transfer of microsatellites within the cereals has been more limited (reference). One advantage of microsatellites derived from ESTs is the potential that they may be more transferable between species. Recently we demonstrated the potential to transfer microsatellite derived from grape ESTs to related species (SCOTT et al. 2000b). We are currently investigating transfer of sugarcane microsatellites derived from ESTs to sorghum and other related species compared with those from the International Consortium for Sugarcane Biotechnology (ICSB).
Microsatellites: The ITEC database was searched for microsatellite sequences and the loci identified are being evaluated for wheat and barley. Preliminary results with wheat indicate low levels of polymorphism for the microsatellites in wheat sequences. This may be due to the types of cDNA libraries represented currently in the ITEC database. We have evaluated 29 SSRs from barley EST sequences. These barley SSRs have also been evaluated in wheat. Most barley SSRs performed well, in contrast to a similar set of wheat SSRs derived from ESTs.
Single nucleotide polymorphisms: Single nucleotide polymorphisms have been detected by analysis of the Genbank DNA database. Comparison of EST sequences from a cDNA library derived from the variety Alexis and the sequences in the database allowed the detection of possible SNP loci. In other cases, over 50 SNP primer pairs have been designed and used to amplify from 10 barley genotypes to reveal potential SNPs. The initial application of these results has been in the design of variety specific assays for use in identification of barley genotypes.
Molecular beacons: The molecular beacon technique was demonstrated to work effectively in the analysis of a transgene in barley (KOTA et al. 1999). This work is being extended to the application of molecular beacons to the detection of SNPs in barley. Variety specific molecular beacons are likely to have wide application in industry.