wheat diploid hexaploid analysis
Discovery of SNPs and genome-specific mutations by comparative analysis of transcriptomes of hexaploid wheat and its diploid ancestors
Plants were grown in greenhouse conditions under natural day light supplemented with artificial source of light. The total RNA was isolated from 4 week seedlings using the RNeasy Plant Mini Kit (QIAGEN). First-strand cDNA synthesis was performed according to SMART cDNA synthesis technology (Clontech Laboratories, Inc.) using modified 3’ SMART CDS Primer II A (5’-AAGCAGTGGTATCAACGCAGAGTACTTTTGT(9)C T(10)VN-3’) and SuperScript III reverse transcriptase (Invitrogen). Double-stranded cDNA was amplified by long-distance (LD) PCR using Advantage 2 PCR Enzyme System (Clontech Laboratories, Inc). Amplification was performed on thermal cycler (Applied Biosystem) with the following PCR parameters: 95°C for 1 min. followed by 16 cycles of 95°C - 15 sec., 65°C – 30 sec., 68°C - 6 min. The quality of double-stranded cDNA was checked on the 1.1% agarose / EtBr gel in 1X TAE buffer and purified with QIAquick PCR Purification Kit (QIAGEN).  The double-stranded cDNA was normalized using TRIMMER cDNA normalization Kit (EVROGEN), which is based on a unique DSN (duplex-specific nuclease) normalization technology and is specifically developed for normalization of cDNA enriched with full-length sequences.
Transcriptomes of two hexaploid wheat (Triticum aestivum) cultivars Jagger and Chinese Spring and three diploid ancestors of the wheat genomes T. urartu (A genome ancestor), Aegilops tauschii (D genome ancestor) and Ae. speltoides (B genome ancestor) were re-sequenced using 454 instrument for inter-varietal SNP discovery and identification of genome-specific divergent mutations differentiating wheat genomes from each other.