Introduction
The Queensland fruit fly, Bactrocera tryoni, is a serious pest that has cost Australian farmers hundreds of millions of dollars due to its destructive nature of Australian horticulture and fruit production. They are widely distributed across the eastern and northern territory of Australia and are known to be Australia’s most difficult and costly biosecurity challenge (Taylor, 2016). Learning more about these flies will help tell us more about their behaviours and adaptations that will help us produce controlled measures to prevent these flies from spreading.
One way to learn more about the phenotypes and traits of these flies is through linkage analysis and genetic mapping of the fruit fly genome, which will help in mapping the positions of genes on chromosomes and lead to the knowledge of improved biological control of the species. Past research by (Zhao, et al., 2003) and (Raphael, et al., 2014) has looked at the genes of traits and came up with the sterile insect technique (SIT) to help control the Queensland fruit flies.
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In genetic mapping there are two types of markers, visible and molecular markers. Visible markers are determined by visible phenotypes such as the white marks on the thorax of the fruit fly while molecular markers are a DNA polymorphism within an individual’s nucleotide sequence that may differ from other individuals within the same population. Visible markers are useful, but molecular markers provide us with finer resolution maps as they fill in the gaps between the number of visible markers (Griffiths et al, 2000). The type of molecular marker used for this experiment is Restriction Fragment Length Polymorphism (RFLP) and it is a useful way of finding DNA polymorphisms by cutting the target DNA with a restriction enzyme (Griffiths et al, 2000).
The aim of the experiment was to use an RFLP (white, RaRb) along with known microsatellites and a phenotypic marker (white marks, wm) to determine if the two genes white and white marks are linked and on which chromosomes they are located on.
Materials and Methods
A male backcross of B. Tryoni was performed to assign the white eye-colour gene and the white marks gene to linkage groups. A polymerase chain reaction (PCR) was then conducted on 20 samples of DNA taken from each fly. The PCR reaction contained 18μl of (67mMTris-HCl, (pH8);16.6mM [NH4]SO4;0.45%TritonX-100;0.2mg/ml gelatin;3 mM MgCl2; 0.5mM nucleotide, dATP, dTTP, dCTP, dGTP) PCR master mix added with 2μl of white 2 and white 5’ext primer mix* (12.5μM each primer) and 3μl of DNA template. Reagents were mixed thoroughly and 2μl of taq polymerase was added to the reaction mix. A control tube was also prepared but with sterile milli-Q water instead of the DNA template. Both tubes were then placed in the thermocycler for approximately 3 hours for a total of 37 cycles between denaturing, annealing and extension. After the PCR reaction was completed, samples were stored at a temperature of -20’C.
On a different day, Half of the PCR product from Day 1 was digested with the restriction enzyme RsaI to determine the genotype of the individual. The other half was run on a gel as an uncut DNA control. The PCR products were spun down in a microfuge before removing 10μl. A RE digest for the PCR product was setup by adding 6μl of MQ water, 2μl of 10X buffer, 10μl of white PCR product and 2μl of RsaI 1.75U/μl and then mixed by giving it a quick touch spin and incubated in a 37C water bath for 1 hour. 2μl of 10 x loading dye was then added and mixed with the digestion reaction and the uncut PCR products. The gel used for electrophoresis was prepared and loaded according to the two tables (see table 5 and 6 in supplementary). The DNA samples were run on the gel until the dye front has travelled just before the next line of wells or the end of the gel (this took about 30 min). The gel was then photographed under long wavelength UV light using a digital imaging system and the images were used to perform a linkage analysis of the results.
Discussion
Linkage analysis in this experiment enabled us to successfully locate that the white marks gene is located on chromosome 2 and it is not linked to the white RFLP gene which was located on chromosome 5. These results came in line with previous research done that mapped the polytene chromosomes of B. Tryoni and found that the Tryoni white gene is linked to chromosome 5 and that the white visible marker gene was on chromosome 2 (Zhao, et al., 2003).
Since the two genes are unlinked and are located on different chromosomes, it is not possible to draw a linking gene map for the two genes of these experiments unless further molecular or visible markers have been studied and located on one of the two chromosomes 2 and 5. Zhao et al (2003) have characterised microsatellites Bt4, Bt14 and Bt32 on chromosome 2, among others (Zhao, et al., 2003). By looking at these markers and those for chromosome 5, a more precise linkage map could be constructed. A larger number of progenies would also be preferable. However, B. Tryoni males do not have recombination during meiosis and so crossing over is not thought to occur, so a smaller sample size can be used to establish linkage (Zhao et al, 2003).
Researching and gene linking analysis of the B. Tryoni genome is important as it leads to further understanding of the behaviours of these fruit flies and how to take the appropriate measures to control them. Genetic research done by Associate Professor Phil Taylor from Macquarie University (Taylor, 2016) and (Raphael, et al., 2014) have lead them to developing the Sterile Insect Technique (SIT) strains that are able to control the reproduction numbers of these flies and limit them. Although SIT is a great technique, it has some disadvantages that include the cost and technical difficulties of establishing sterile males (Raphael, et al., 2014).
Experiments such as the one performed further widens the knowledge on the genome of these flies and how each gene is linked to another, and that way more and more strategies to control these flies are explored and implemented to help save the economy of the horticulture in Australia.
References
- Griffiths AJF, Miller JH, Suzuki DT, et al. (2000). An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; Mapping with molecular markers.
- Raphael, K. A., Shearman, D. C., Gilchrist, A. S., Sved, J. A., Morrow, J. L., Sherwin, W. B., … Frommer, M. (2014). Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique. BMC genetics, 15 Suppl 2(Suppl 2), S9. doi:10.1186/1471-2156-15-S2-S9
- Taylor, P., 2016. The Queensland Fruit Fly, Sydney: Maquarie University .
- Zhao, J. T., Frommer, M., Sved, J. A. & Gillies, C. B., 2003. Genetic and Molecular Markers of the Queensland Fruit Fly, Bactrocera tryoni. Journal of Heredity, 94(5), pp. 416-420.
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