Construction of a rye (Secale cereale L.) reference transcriptome

As a test case for building a complete reference transcriptome for a eukaryotic species with a large un-sequenced genome, more than 5.6 million high quality 454 pyrosequencing cDNA reads from rye (Secale cereale) were generated and assembled. A novel post de novo assembly sequence analysis program, BLAST-based post-assembly process (BbPAP), was designed to eliminate redundancy, maximize contig length, and correct erroneous assemblies by recognizing and eliminating alternatively spliced transcripts and error-rich termini regions while integrating EST and genome sequence information from related species. A rye reference transcriptome consisting of 56,091 sequences with an average median length value of 1,301 nucleotides was obtained. It includes 41,567 coding loci, covering more than 90% of the hypothetical gene loci. More than 80% of the identified genes have complete coding regions representing the most complete gene index available in any Triticeae species. In addition, we identified that approximately 20% of the genes had putative alternative splicing sites. A minimum expansion of rye genes of 17% was calculated based upon comparison with orthologous single copy genes from Brachypodium. 4,461 novel rye or Triticeae-specific transcripts were identified and half of these were subjected to purification selection thus indicating their important functions in the formation of the Triticeae species. none provided