Development and functional assessment of EST-derived 2RL-specific markers for 2BS.2RL translocations

ESTs-derived markers are useful for comparative genomic analysis and can also serve as phenotype-linked functional markers. Here, we report the development of EST-derived 2RL-specific markers and the evaluation of the possibility of functional assessment of markers tagging 2RL, which carries Hessian fly resistance genes (loci). To identify transcripts specific to 2RL, unigene sequences in combination with wheat progenitor genomes were used. Total 275 contigs mapped to the long arms of homoeologous group 2 chromosomes were downloaded. To obtain a cluster corresponding to each of the wheat 275 contigs, unigene sequences of wheat, rice, barley, and rye were pooled for cross-species clusters. Out of 275 clusters examined, it was possible to design 112 cross-species primer pairs for genome-specific amplifications. Out of 112 cross-species primer pairs, 45 primer pairs (40%) produced amplicons from at least one species (three wheat progenitors or rye). Among the 45 contigs, 73% were associated with one of known functions and 82% of the contigs associated with known functions were also associated with one of the GO categories. On the basis of the oligonucleotide sequence alignment of each of 45 genome-specific amplifications, 21 amplifications (47%) were suitable for designing RR genome-specific primers, which are specific to translocated rye chromatin 2RL. Six primer pairs (13%) successfully produced amplicons in the 2BS.2RL translocation lines and not in the non-2RLs. Functional assessment of one of the 2RL-specific markers, NSFT03P2_Contig4445, was performed on Hessian fly infested NILs. Under Hessian fly infestation, significantly high expression of a gene tagged by a 2RL-specific marker (NSFT03P2_Contig4445) was observed 1 day after infestation. EST-derived 2RL-specific marker development from this study provides a basis for the development of ESTs-derived markers for detecting wheat-rye translocations. In addition, these markers could be employed in elucidating functional analysis of genes on 2RL.