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Transcriptomics in Coffee

Despite the tremendous economic values of coffee, our understanding of its genome is still very poor. The main obstacles are the narrow genetic base of the main cultivars, the autogamous reproductive strategy of the plant, the long generation period and the relatively limited resources allocated to the genetic/molecular studies. In view of the above, we focused our research onto two major objectives: first of all we want, to identify and exploit the largest possible number of expressed genes in the two tissues particularly involved in resistance response. Secondly, to develop the trascriptomics of resistance response through microarray analysis of Expressed Sequence Tags (ESTs).

This is currently being done at University of Trieste, Italy

Could it be a milestone?

The ESTs are single-pass sequences of cDNA clones randomly chosen from a library, they can be used for variety characterization as well as for expression studies. We recovered a total of 2069 ESTs from 3'-end specific cDNA libraries of roots and leaves of C. arabica. The amplification of some of the ESTs on arabica varieties resulted in identification of 3 single nucleotide polymorphic (SNPs), and several heterozygous loci. These SNPs are under development as diagnostic markers.

The DNA microarray technology is a most promising and dynamic approach for functional genomics. The identified ESTs have been assembled into 1520 contigs (900 from roots and 620 from leaves). A set of 48 gene fragments with an NBS-LRR domain have been also sequenced from two genomic library enriched for gene linked to the resistance to several pathogen. The DNA inserts of cDNA were PCR amplified, purified and spotted on coated glass slides. Total RNA was isolated (400 µg) and the mRNA was purified (1 µg) from roots and leaves of three plants of Bourbon VD before and after treatment with 0.125 mg/ml of Bion 50 WD® (acibenzolar-s-methyl, Syngenta) for one day. Bion elicits systemic acquired resistance (the salicylate pathway). The first strand cDNA has been labelled with fluorochromes and a competitive hybridisation was carried on. Quantitation and normalization of data has been obtained by spiking the target mRNA with known concentration of control RNA. Some unknown cDNA fragment results over-expressed after treatment and we are now working in full-length cDNA sequencing.