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Title: Development of molecular genetic markers from a cDNA subtraction library of Frosty Pod inoculated cacao

item Kuhn, David
item Macarthur, Heather
item Nakamura, Kyoko
item Borrone, James
item Schnell Ii, Raymond
item Brown, James

Submitted to: Proceedings of the XVth International Cacao Conference
Publication Type: Proceedings
Publication Acceptance Date: 10/9/2006
Publication Date: 12/31/2006
Citation: Kuhn, D.N., Macarthur, H.C., Borrone, J.W., Schnell Ii, R.J., Brown, J.S., Johnson, E., Phillips-Mora, W. 2006. Development of molecular genetic markers from a cDNA subtraction library of Frosty Pod inoculated cacao. Proceedings of the XVth International Cacao Conference.

Interpretive Summary: Theobroma cacao, the source of cocoa beans for chocolate, is an important tropical agriculture commodity that is affected by a number of fungal pathogens, including frosty pod (Moniliophthrora roreri H.C. Evans, Stalpers, Samson & Benny). We are trying to find molecular genetic markers that are linked to disease resistance to frosty pod in Theobroma cacao to aid in a marker assisted selection (MAS) breeding program for cacao to ensure a reliable supply of cacao for the US confectionary industry. A novel approach to identify such markers is to find the genes that are turned on (up-regulated) or turned off (down-regulated) after the plant is attacked with the pathogen. We prepared four cDNA subtraction library from cacao pods that had been inoculated with frosty pod spores to identify differentiallly regulated genes. The library that had the most up-regulated genes was from a cultivar resistant to frosty pod. We are now determining if any of these genes can be used as a genetic marker for frosty pod resistance. Our results are important to scientists trying to understand the mechanism for disease resistance and, eventually, to cacao farmers who will benefit from superior disease resistant cultivars produced through our MAS breeding program.

Technical Abstract: We have been employing a candidate gene approach to identify molecular markers associated with disease resistance in Theobroma cacao. Candidate genes can be turned into molecular markers using single strand conformation polymorphism (SSCP) analysis. As a novel approach to identifying genes associated with resistance to Moniliophthora roreri, the causal agent of frosty pod disease, we have created subtracted cDNA libraries from pathogen-inoculated cacao pods. We created the subtraction libraries by preparing cDNA libraries from pods of a resistant cultivar of cacao (UF273) and pods of a susceptible cultivar of cacao (Pound 7) that were either inoculated with the frosty pod pathogen (Moniliophthora roreri) or uninoculated. Pods were inoculated and lyophilized in June, 2004 at the CATIE research station in Turrialba, Costa Rica. RNA was isolated and reverse transcribed into cDNA at the USDA ARS SHRS Plant Science laboratory in Miami, Florida, USA. For each cultivar, a forward and reverse subtraction library was prepared. The forward subtraction has only cDNAs from genes that were induced by inoculation. The reverse library has only genes that were down regulated by inoculation. All cDNAs common to both the forward and reverse libraries for each cultivar are removed (subtracted), which reduces the cost in time and resources to identify genes regulated by pathogen inoculation. The UF273 forward and reverse libraries have approximately 1800 clones for each. The Pound 7 forward and reverse libraries have approximately 900 clones for each. Preliminary sequencing of about 200 clones from each library has identified the UF273 forward library as containing the greatest number of unique sequences. The majority of these sequences have been identified by TBLASTX search of the NCBI nr database and are similar to genes that are known to be upregulated in disease resistance in other plants. Sequencing of all clones from all libraries is near completion. We have observed sufficient redundancy in all libraries to allow us to identify single nucleotide polymorphisms (SNP) in the sequences unique to each library, which we will use to develop these loci as genetic markers. These SNPs will be mapped in a UF273 x Pound 7 population that is segregating for resistance to frosty pod to determine association with QTL for frostypod resistance.