SNP discovery outline

This online database contains information from a project at the James Hutton Institute to mine wheat and barley genes for SNPs which were mapped in barley crosses. A synopsis and further details about the project can be seen here. An online abstract of the published paper can be seen here.

Selection of the genes

We focused on genes associated with abiotic stress in barley. The reference point for each gene was an EST unigene from the HarvEST Barley Assembly 21 which was used to design Affymetrix probe sets for the Barley1 GeneChip.

Barley germplasm

A selection of EST unigenes was analyzed for sequence polymorphisms in 8 diverse barley genotypes: OWB D, OWB R, Steptoe, Morex, Lina, HS92, Optic and Golden Promise. These include parents of three doubled haploid mapping populations facilitating linkage mapping of genes and represent wide range of cultivated barley germplasm from Europe and North America, as well as a wild barley accession.

Primer design, PCR and sequencing

Primers were designed with Primer3 software targeting the 3' ends of unigenes. PCR was carried out on a panel of 8 genotypes, PCR products were treated with exonuclease and shrimp alkaline phosphatase and single-pass sequencing was done with the same primers as PCR using Applied Biosystems BigDye terminator kit.

Sequence analysis

Base calling and sequence assembly was done with phred and phrap . Mutations were identified using polyphred and confirmed by manual examination of sequence assemblies in consed .

Polymorphism database

Mutations identified by polyphred and the contig alignments were processed using custom-made Perl scripts and entered into a mySQL database.

Reference

Rostoks N, Mudie S, Cardle L, Russell J, Ramsay L, Booth A, Svensson JT, Wanamaker SI, Walia H, Rodriguez EM, Hedley PE, Liu H, Morris J, Close TJ, Marshall DF, Waugh R (2005)   "Genome-wide SNP discovery and linkage analysis in barley based on genes responsive to abiotic stress."   Mol. Genet. Genomics 1-13