Analysis of wheat SAGE tags reveals evidence for widespread antisense transcription.

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat. RESULTS: Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression. CONCLUSION: Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.

Molecular markers in breeding for virus resistance in barley.

The soil-borne barley yellow mosaic virus disease (BaMMV, BaYMV, BaYMV-2) and the aphid-transmitted barley yellow dwarf virus (BYDV) are serious threats to winter barley cultivation. Resistance to barley yellow mosaic virus disease has been identified in extensive screening programmes and several recessive resistance genes have been mapped, e.g. rym4, rym5, rym9, rym11, rym13. In contrast to barley yellow mosaic virus disease, no complete resistance to BYDV is known in the barley gene pool, but tolerant accessions have been identified and QTL for BYDV-tolerance have been detected on chromosomes 2HL and 3HL. The use of resistance and tolerance in barley breeding can be considerably improved today by molecular markers (RFLPs, RAPDs, AFLPs, SSRs, STSs, SNPs), as they facilitate (i) efficient genotyping and estimation of genetic diversity; (ii) reliable selection on a single plant level independent of symptom expression in the field (iii) acceleration of back crossing procedures; (iv) pyramiding of resistance genes; (v) detection of QTL and marker-based combination of positive alleles; and (vi) isolation of resistance genes via map-based cloning.