Development of microsatellite enriched libraries in several tropical species

The use of microsatellite markers for diversity studies and mapping has been undertaken for several tropical species including sugarcane, rubber tree, cocoa, oil palm, and coconut palm. Three different small insert microsatellite enriched libraries have been constructed for each of the above species. These libraries include one for each of the tetra nucleotide repeats GA and GT and a mixed library enriched for the tri and tetra repeats CAA, ATT, ACC, GCC, GAA, CATA and GATA. Initial screening of the libraries was performed by PCR amplification of the cloned inserts followed by Southern blot hybridization. The degree of enrichment for the libraries varied by the type of microsatellite as well as by species. The percentage of enrichment ranged from 15% to >65%. Representative clones from each of the libraries was sequenced to confirm the presence and determine the average size of the microsatellite sequences. The type and size of microsatellites found varied among the species particularly those from the mixed library. Unique primers have been designed from the regions flanking the microsatellite sequences and then used for PCR amplification. The results of each amplification were analyzed first by agarose gel electrophoresis to confirm that a product of the expected size was produced. Successful primer pairs were then used on progeny populations for mapping and on cultivars for diversity studies. Amplification products were then analyzed by ethidium bromide staining of 4% agarose gels or on acrylamide gels followed by autoradiography. A project sponsored by the Genoscope, Evry, France is producing sequence data for 250 additional clones for each of the above species enabling us to increase the number of potential markers for each species. (Texte intégral