An EST survey of the sugarcane transcriptome.

Its large genome and high polyploidy makes sugarcane (Saccharum spp.) a singularly challenging crop to study and improve using genetic approaches. To provide large numbers of functionally characterized candidate genes that might be tested for direct association (rather than distant linkage) with economically important traits, we sequenced the 5' ends of 9,216 clones from three cDNA libraries (apex, leaf and mature internode), representing 3,401 non-redundant sequences. About 57% of these sequences could be assigned a tentative function based on statistically significant similarity to previously characterized proteins or DNA sequences. Another 28% corresponded to previously identified, but uncharacterized, sequences. Some of the remaining unidentified sequences were predicted to be genes which could potentially be new to plants or unique to sugarcane. Comparisons of the sugarcane ESTs to a large sorghum EST database revealed similar compositions of expressed genes between some different tissues. Comparison to a detailed Arabidopsis protein database showed some highly conserved sequences, which might be useful DNA markers for pan-angiosperm comparative mapping. These EST sequences provide a foundation for many new studies to accelerate isolation of agronomically important genes from the cumbersome sugarcane genome.

Inheritance and segregation of virus and herbicide resistance transgenes in sugarcane.

Transgenic sugarcane parents containing multiple copies of herbicide resistance ( bar) and Sorghum mosaic virus (SrMV) resistance ( hut) genes were crossed with non-transgenic sugarcane varieties. Segregation of the transgenes in the progeny was determined using Southern blot analysis; herbicide resistance and SrMV resistance were assessed using bioassays. The segregation data were used to infer linkage relationships between transgenes in the parent plants. In two of the parents, all transgene insertions were linked in one position in the genome, although some recombination between insertion events did occur. In the third parent, insertion had occurred in two independent, unlinked loci. Analysis of progeny of this parent indicated that rearrangement or mutation occurred in both loci, resulting in non-parental transgene DNA fragments in some progeny. Most transgenic progeny containing the bar gene showed resistance to herbicide. SrMV inoculation indicated that a fairly high proportion of the transgenic progeny showed susceptibility. As the post-transcriptional gene silencing mechanism responsible for the virus resistance phenotype may be reset during meiosis, phenotypic screening of older plants may be a more reliable indication of virus resistance than screening young seedlings. This is the first report of transgene segregation in sugarcane, and we have demonstrated that transgenic sugarcane parents showing stable inheritance of transgenes can be effectively used in breeding programs.