Generation of microsatellite markers in the medfly, Ceratitis capitata, and their use in physical and genetic mapping

Full text: Microsatellites are tandem repeats of short (2-5) nucleotide motifs, which are present in high numbers in most eukaryotic genomes. They have been used for high-density linkage maps because they are highly polymorphic and widely distributed throughout the genome, readily assayable by polymerase chain reaction (PCR) and applicable to diploid crosses using any polymorphic strain. Eight different small-insert genomic libraries were constructed from genomic DNA extracted from ∼100 flies of the wild type strain, Benakeion, digested separately with AluI, RsaI, MboI, HpaII or Sau3A and cloned into pBS or pUC18 plasmid vectors. Clones containing microsatellites were identified by filter hybridization mainly with radiolabeled (TC)15 and (TG)15 oligonucleotide probes. Following the DNA sequencing of positive clones a total of 132 microsatellite-bearing clones were identified. A number of clones were found to contain more than one distinctive tandem repeat, thus raising the number of microsatellites to 223. The most common microsatellite motif was the dinucleotide (TG)n/(CA)n occurring in 105 out of the 223 microsatellite loci. The physical mapping of the microsatellite clones to the medfly polytene chromosomes was performed using fluorescence in situ hybridization (FISH). The distribution of mapped loci was neither analogous to the size of the chromosomes nor uniform throughout each chromosome. For genetic mapping, pairs of primers were designed based on unique sequences flanking the repeat array. These pairs of primers were used to PCR amplify the corresponding genomic fragment. The primer pairs were then screened against genomic DNA from the Benakeion strain and those ones, which amplified the expected size, were in turn used for the genotyping of F2 progeny in a number of single pair crosses. Linkage analysis was performed using the Join Map 3.0 computer package and the resulting linkage groups were assigned to the corresponding chromosomes using as landmarks the physical localization of the respective microsallite loci. Integrated genetic maps were constructed for the four out of the five autosomes using the microsatellite loci and morphological markers. These results constitute the basis towards the construction of the medfly microsatellite map, which do not however cover the entire genome. Taking into account the genome size of C. capitata as well as the non-random distribution of the so far analysed micosatellite loci, more loci are needed to achieve this goal. (author)