Plant & Animal Genomes XVII Conference

January 10-14, 2009
Town & Country Convention Center
San Diego, CA

Use Of Normalized cDNA Libraries For Enrichment Of Non-Redundant EST Sequences And Construction Of A cDNA Array For Functional Analysis Of The Coffee Transcriptome

Carmenza Góngora¹ , Alvaro Gaitán¹ , Pilar Moncada¹ , Marco Cristancho¹ , L. Fernando Rivera¹ , Carlos E. Orozco¹ , Robin Buell^3,4 , Gabriel Cadena¹ , Herb Aldwinckle² , Marcela Yepes²

¹  Colombian National Coffee Research Center -CENICAFE- Chinchiná, Caldas, Colombia

²  Department of Plant Pathology, Cornell University, Geneva, NY 14456, USA

³  The Institute for Genomic Research (TIGR), 9712 Medical Center Drive, Rockville Maryland 20850, USA

⁴  Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA

A collaborative research project between the Colombian National Coffee Research Center (CENICAFE), Cornell University, and the Institute for Genomic Research (TIGR) co-funded by the Colombian Coffee Growers Federation, the Colombian Ministry of Agriculture, and the US National Science Foundation focused on the construction of three coffee normalized cDNA libraries: one for the allotetraploid Coffea arabica (mixed tissue), and two normalized libraries for the diploid species C. liberica (leaf and seeds). Normalized libraries enriched for non-redundant expressed sequence tagged (EST) sequences were used in the fabrication of a coffee c-DNA array for functional analysis of the coffee transcriptome. The coffee microarray includes 33,263 cDNAs (19,074 correspond to C. arabica and 14,189 to C. liberica) and has been used to obtain expression profiling data, using RNA populations from coffee plants from genotypes with differential response to biotic stress (rust and coffee berry borer) for analysis of pathways and novel candidate genes that could be manipulated to enhance resistance. The generation of normalized libraries allowed a broader survey of the C. arabica and C. liberica transcriptome by enriching for rare mRNAs including transcripts from five different tissues (leaf, roots, seeds, flowers, and callus). Normalization generated a 2X enrichment in unigenes reducing redundancy and increasing array density. Quality control during amplification and sequencing allowed to generate longer cDNAs that were spotted directly on the screening array. The coffee microarray is also being used to identify genes affecting other traits of importance in coffee production.