|Winterstein, Michael - Mike|
|Schnell ii, Raymond|
Submitted to: Theoretical and Applied Genetics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/17/2002
Publication Date: 3/21/2003
Citation: Kuhn, D.N., Heath, M.A., Winterstein, M.C., Wisser, R.W., Meerow, A.W., Brown, J.S., Lopes, U., Schnell II, R.J. 2003. Resistance gene homologues in Theobroma cacao as useful genetic markers. Theoretical and Applied Genetics. 107:191-202. Interpretive Summary: Plant disease resistance genes can be grouped by families and are clustered within the genome. One class of plant resistance genes has a nucleotide binding site motif and leucine-rich repeat (NBS/LLR). Using the polymerase chain reaction we were able to amplify and characterize 74 unique resistance gene NBS/LLR homologues from Theobroma cacao. Cacao beans, the seeds of T. cacao, are used in the manufacture of chocolate. These 74 genes could be placed into 11 groups based on DNA sequence similarity. Utilizing another technique that separates DNA into single stranded molecules we were able to detect sequence differences that allowed us to utilize these resistance gene homologues as DNA markers. We were able to map several of the resistance gene gomologues in an experimental population. The ability to utilize these a moleular markers has significant implications for locating disease resistnce genes in plants.
Technical Abstract: Resistance gene homologues (RGH) sequences have been developed into useful genetic markers for marker-assisted selection (MAS) of disease resistant Theobroma cacao. a plasmid library of amplified fragments was created from seven different cultivars of cacao. Over 600 cloned recombinant amplicons were evaluated. From these, 74 unique RGH were identified that could be placed into 11 categories bsed on sequence analysis. Several of these categories contain multiple loci. Primers specific to each category were designed. The primers for a single RGH class amplified fragments of equal length from the seven different cultivars used to create the library. However, these fragments exhibited single strand conformational polymorphism (SSCP), which allowed us to determine their segregation in an F2 population of T. cacao. As SSCP can be efficiently perfromed on our automated sequencer, we have developed a convenient and rapid assay for RGH alleles.