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Title: Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.)

item Glynn, Neil
item Comstock, Jack
item Sood, Sushma
item Dang, Phat
item Chaparro, Jose

Submitted to: Pest Management Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/2/2007
Publication Date: 1/1/2008
Citation: Glynn, N.C., Comstock, J.C., Sood, S.G., Dang, P.M., Chaparro, J.X. 2008. Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.). Pest Manag. Sci. 64:48-56.

Interpretive Summary: Sugarcane production in Florida is affected by diseases, and the only viable form of protection against many sugarcane diseases is through varietal resistance. The greatest losses due to disease occur when resistance breaks down due to more virulent forms of the pathogen. As a result, breeding for disease resistance is an on going challenge in the production of new sugarcane varieties. Typically the time from the initial cross to the release of a commercial variety is 10 plus years, this time could be reduced significantly through the early identification of progeny that have inherited gene(s) for disease resistance. The challenge is to identify such genes (and/ or alleles). Plant disease resistance genes share common structural features between often distantly related species of both dicot and monocot plants. The most common of these contain the Nucleotide Binding Site and Leucine Rich Repeat (NBS-LRR) domains. Kinase genes have also been shown to be involved in the expression of disease resistance and proximal to NBS-LRR resistance genes. The isolation and identification of disease resistance genes can be lengthy and expensive however the isolation of sequences that show similarities to known resistance genes (resistance gene analogues RGAs) is more straightforward. Here we report the isolation of NBS-LRR and kinase gene sequences from a sugarcane clone known to be resistant to YLS and moderately resistant to brown rust caused by Puccinia melanocephala. Some 1512 sequences were isolated which and assembled into 134 separate contigs, 20 showed homology to NBS-LRR resistance genes and 7 showed homology to kinase genes. The sequences will now be examined for polymorphism and associations made with the phenotypic expression of resistance. These are first steps towards developing useful molecular markers for disease resistance for use in our on going efforts in developing disease resistant sugarcane varieties.

Technical Abstract: Resistance gene analogues (RGAs) offer great potential to breeding for disease resistance through marker assisted selection, either as closely linked markers or through candidate gene approaches. Many R-gene sequences contain kinase domains and indeed kinase genes have been reported as being proximal to R-genes making kinase analogues an additionally promising target for the development of markers for disease resistance. We used previously reported degenerate primers targeting three conserved motifs within the nucleotide binding site-leucine rich repeat (NBS-LRR) class of resistance genes and two that targeted conserved motifs within kinase genes to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. This clone was characterized as resistant to yellow leaf syndrome and moderately resistant to brown rust infection caused by Puccinia melanocephala. Amplification products were purified, cloned and 1512 positive clones sequenced, these were assembled into 134 contigs of between 2 and 105 sequences. Comparison of the contig consensuses to the NCBI sequence database using BLASTx showed 20 had sequence homology to NBS-LRR RGAs and 7 to kinase gene accessions. Alignment of the deduced amino acid sequences of the consensus sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. This is the first step towards exploiting the potential of RGA and kinase analogue sequences in breeding for disease resistance through marker assisted selection.