A cDNA subtraction approach to isolate male-specific genes from Ceratitis capitata : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

Abstract

The Mediterranean fruit fly, Ceraritis capitata (medfly), is a significant world wide agricultural pest. Sterile Insect Technique (SIT) is a biological method that has been used to control medfly successfully in several parts of the world for around two decades. SIT involves the release of sterile insects into wild populations which, due to sterile matings, lead to a reduction in the size of the wild population. The effectiveness of this technique is significantly increased when only sterile males are released. This can be achieved by using sexing strains, but these strains are prone to breakdown as a result of recombination leading to the disassociation of the selection gene from the Y chromosome. An alternative system, that could be more robust, would involve the control of the expression of the male determining gene. The aim of this thesis was to identify the male determining gene of medfly by creating subtracted cDNA libraries enriched for male-specific transcripts. Subtracted libraries were made by subtractive suppression PCR, using the ClonTech cDNA subtraction kit. The libraries were screened by Southern hybridisation analysis using male and female total cDNA probes. Only one clone appeared to display a bias toward male-specific hybridisation, but this was found to be a result of unequal transfer of DNA. A selection of clones were individually used to probe membrane bound genomic DNA. These hybridisation analyses indicated a general lack of male-specific enrichment. In addition to this, sequence analysis of a selection of clones revealed a number of mitochondrial gene fragments, showing that there had been insufficient subtraction. As results indicated that the creation of subtracted, male-specifically enriched libraries had been unsuccessful another approach to the identification of the male determining gene was attempted. Genomic DNA was screened with an Sxl probe, under low stringency hybridisation conditions, to identify Y-linked encoding RNA binding proteins distantly related Sxl, which could represent the male determining gene. This screen showed that there were no male-specific RNA binding proteins, of the SXL family in medfly