Trinucleotide Repeats as Bait for Vectorette PCR: A Tool for Developing Genetic Mapping Markers

Trinucleotide repeats are common within gene coding regions and could serve as beacons to locate genes. Five of the most common trinucleotide repeats in an Actinidia (kiwifruit) expressed sequence tag (EST) database were found to be (ACC)₄, (CAC)₄, (CCA)₄, (CTC)₄, and (TGG)₄. These repeats, with or without an artificial 5′-end tail, were tested by vectorette PCR against genomic DNA from Actinidia chinensis. Eighty-nine randomly selected clones showed an average insert size of 383 bp, with a maximum of 1,151 bp and a minimum of 78 bp. Two-thirds of the clones contained the artificial tail attached to the trinucleotide, showing a slight advantage of possessing such a tail during annealing and amplification. The sequences were searched against the Actinidia EST database and GenBank. Of the 89 clones, 33 had a significant hit (expect value < e⁻¹⁵). Twenty-four of those clones matched an Actinidia EST. Twenty-one clones contained one or more simple sequence repeats. This methodology can be applied by conventional cloning and sequencing methods or by high throughput pyrosequencing technologies to develop genetic markers and also for gene mining in species with little or no genetic/genomic resources.