DNA sequences differentially represented in males and females of the oriental fruit fly Bactrocera dorsalis

The objective of this dissertation is the isolation of DNA sequences that are differentially represented in males and females of the Oriental fruit fly Bactrocera dorsalis, specifically by initiating a molecular characterization of Y chromosome sequences in this species. Cytological observations have established the presence of a diminutive Y chromosome in B. dorsalis males. To isolate DNA sequences from the Y chromosome, a special method of genomic DNA isolation known as Representational Difference Analysis (RDA) was utilized to obtain DNA sequences unique to the B. dorsalis male genome. Genomic DNA from B. dorsalis males served as the "tester" DNA and female genomic DNA as the "driver" DNA. Six distinct RDA products were obtained following two complete rounds of DNA hybridization and difference enrichment via the Polymerase Chain Reaction (PCR). One ofthese products (RDA product 1) was used to isolate a genomic DNA clone (3.1a) from a B. dorsalis male genomic DNA minilibrary. This sequence shows similarity to the reverse transcriptase of R1 retrotransposable elements. The presence of R1 elements in the Tephritid insects has heretofore been undescribed, although these elements have been previously described in the genomes of other Dipteran species. Oligonucleotide primers for PCR were designed for the 3.1a clone. These primers consistently produce different amplification patterns in PCRs ofgenomic DNA from B. dorsalis males vs. females. Amplification using male genomic DNA produces 325 bp and 2.6 kb products while only a 2.6 kb product is obtained from female DNA. The amplification products obtained with these primers are also produced in PCRs of genomic DNA from B. dorsalis embryos and third instar larvae, suggesting the ability of this method to infer sex at pre-adult stages ofthe B. dorsalis life cycle. Similar amplification products have also been obtained in other Bactrocera species. Both the 325 bp male PCR product and the 2.6 kb products have regions of sequence similarity to R1 elements. The 2.6 kb product contains a putative 1.7 kb open reading frame (ORF) encoding 583 amino acids. Three amino acid motifs found in Drosophila R1 element reverse transcriptases are present in comparable locations within the hypothetical ORF product. Both of these sequences are also repetitively represented in the B. dorsalis male and female genomes. However, the 325 bp male product produces some bands that are male specific when used as a probe for Southern blots of B. dorsalis male and female genomic DNA. The amplification pattern produced by the 3.1a primers is consistent with what would be expected if the 2.6 kb and 325 bp PCR products originated from the B. dorsalis X and Y chromosomes, respectively. Thus, the cloned male-specific sequence recovered here is potentially useful both as a gateway into the relatively uncharacterized B. dorsalis Y chromosome and as a tool for the characterization of other aspects of the B. dorsalis genome