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Development of Coffee Microsatellites

Genetic improvement of coffee, can particularly be triggered using molecular marker approaches. Molecular Breeding based on polymorphic DNA markers/techniques offers possibilities to overcome these constraints but require development and mapping of coffee specific DNA markers and molecular linkage maps. In this context, it is the small tandem repeat based microsatellite markers that have proven to be most desirable in various genetic studies on germplasm characterization and linkage analysis due to their codominant nature, stability, abundance, sensitivity, ease and speed of analysis, minimal sample requirements and suitability for automation.

Microsatellite Validation

Of the repeat positive clones, 17 primers were validated as true SSR markers for polymorphism information content (PIC) and cross-species specificity using panels of elite arabica genotypes (29 improved selections) and 17 Coffea species. The analysis revealed few alleles (av. 2.6) coupled with low PIC (av.0.4) in arabica genotypes but more number of alleles (av. 8.6) and high PIC (0.8) between the species. The study thus provide the large number of coffee specific microsatellite markers described till to date, that can be used to investigate the level of genetic variation of coffee germplasm, for evolutionary/taxonomic studies and development of molecular linkage map of coffee..

Microsatellite Developement

For the purpose, we have constructed a small insert, partial, genomic library comprising about 50,000 clones having inserts of 0.8-1.0 kb, from the total DNA of an elite genotype of Coffea arabica var. HDT (Hibrido De Timor). The latter is a putative natural interspecific hybrid of C. arabica and C. canephora and is used as a universal donor of rust resistance. High density filters representing around 12,000 of these clones were screened through Southern hybridization with 9 different synthetic oligonucleotide repeat probes viz., (CA)15, (GA)15, (CAA)10, (GGT)10, (ATT)10, (AGG)10, (AGA)10, (ACT)10 and (CATA)8. Based on the hybridization signals, a large number of putative repeat-positive clones were identified of which 288 were fully sequenced for both the strands. Of these 143 clones were found to carry the repeats suggesting an estimated occurrence of ~3.37x104 repeats per haploid genome of C. arabica. Among various repeats, (AT) n repeats were found to be the most abundant followed by (GA)n, (CA)n and (ATT)n that were estimated to be ~ 2.6x104, 3x103, 2.3x103, and 1.95x103 respectively, in the arabica genome. These estimates corroborates well with our dot-blot based comparative hybridization results on relative abundance and distribution of different repeat motifs in the genome of different coffee species.

This work was done at Center for Cellular and Molecular Biology, India. Click on the Publication section to view the recent article.

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