Genome-Specific Primer Design for Wheat SNP Discovery

Conserved PCR primer design:
The conserved PCR primers are designed to amplify about 1,000 bp of wheat genomic sequence. The
nested sequencing primers will be simultaneously designed for each hypothetical amplicon.

PCR amplification and sequencing of conserved STSs and design of genome specific primers:
The targeted genomic STSs will be PCR amplified from genomic DNAs of 3 accessions each of T. urartu,
Ae. speltoides, and Ae. tauschii. Genetic distances have been determined among at least 50 accessions
of each species (see proposal for details). These genetic distances will be used to select the diverse
lines within each species. The relationship between the wheat B genome and the genome of Ae.
speltoides, its putative ancestor, is uncertain, and there is no guarantee that the primers designed on the
basis of Ae. speltoides sequence will work in wheat. Since the location of a targeted locus will be known
in most cases, the B-genome sequence can be often obtained using DNA of an A- or D-genome
nullisomic B-genome tetrasomic line (N-T) for the chromosome on which the locus is located. For
example, for a locus on homoeologous group 1 chromosomes, DNAs of N1A-T1B and N1D-T1B will be
used for PCR amplification. Finally, one accession of T. turgidum dicoccoides will be included. The PCR
product will be identified by gel electrophoresis, excised, and purified on spin columns using the Magic
PCR Purification Kit (Promega) or an analogous commercial product.

Centralized sequencing of conserved STSs will be implemented.  The concentration of the amplicons in
plates will be normalized using the Tecan Genesis 150 robot and the normalization program that is
available for the instrument. The nested sequencing primers will be selected from the primer database
and ordered. The plates with normalized amplicons and sequencing primers will be mailed for centralized
sequencing.

To produce a genome-specific primer pair, one genome-specific primer will be combined with one of the
conserved primers. The genome specificity of each primer pair must be individually scrutinized against
wheat N-T lines. The N-T test is used to verify the location of targeted STSs, or identify their locations for
those amplified by chance from a paralog.