A molecular marker is a DNA polymorphism within a single nucleotide sequence that can distinct from other individuals within the identical population. In these makers, a visible marker causes a change in phenotype. However, these markers do not create an observable difference in phenotype, but both markers have similar characteristics. Moreover, both visible and noticeable markers can be utilized and generate a linkage map with far more intention than a map make use of phenotypic markers alone (Griffiths et al., 2005).
Cutting the target DNA along a restriction enzyme is a beneficial technique of discovering DNA polymorphisms. Moreover, each of the restriction enzymes has a particular identification site, and distinction site between individuals will lead to variable restriction fragment lengths follow by digestion. Thus, determining the order of the DNA is not required. Furthermore, Restriction Fragment Length Polymorphisms (RFLPs) method is used to detect the molecular markers (Griffiths et al., 2008)
Bactrocera tryoni, which is also known as the Queensland Fruit Fly, was focused in this experiment. Bactrocera tryoni is the most significant pest of horticulture in Queensland, NSW and Victoria (Genetics and Genomic 2001 Lab Manual, 2019). It is essential to study this experiment and have additional knowledge about genome because, in the future, genome could guide to improve dealing with the biological species. Many articles have been published on the genome of Bactrocera tryoni, the Queensland Fruit Fly by The University of Sydney's Fruit Fly Research Centre including Frommer & Bennett, 1997; Kinnear et al., 2006; Zhao et al., 1998; Zhao et al., 2003; Zhao & Frommer, 2006.
The main aim of this experiment was to use a visible marker (white markers or wm for short) and molecular markers along with a microsatellite and an RFLP to locate the white gene of Bactrocera tryoni on the chromosome map. During meiosis, male Bactrocera tryoni do not present crossing over, meaning the genes on the same chromosome will be passed on, and the investigation of the linkage can be done along with a smaller number of offspring (Zhao et al., 2003).
Materials and Methods
In this experiment, male backcross of Bactrocera tryoni was carried out to allocate the white gene to linkage groups. Furthermore, a fragment of the white gene of the 16 individual G2 samples was amplified by utilizing the Polymerase Chain Reaction (PCR). Moreover, the product of the PCR was dissolved using the restriction enzyme Rsal, and gel electrophoresis were carried out with the digestion's products. To calculate the fragment size, a lane on the gel consisting of pUC19 standard was used. Gel electrophoresis of the undigested of PCR product was performed as a control. Finally, examine and investigate the distance migrated by the DNA bands and gel photo.
An RFLP at Rsal sites in the white gene has been characterized and found between the gene of the intron 5 (Frommer & Bennett, 1997). With the Bt2 microsatellite linkage analysis, the white gene Bactrocera tryoni was favourably situated on chromosome 5, which is in line with the past cytogenetic work that outlined the Bactrocera tryoni's polytene chromosomes (Zhao et al., 1998). In this experiment, the marker has been titled as Rwhite, which is the same marker that we have investigated. In the previous studies of the morphological marker, the wm gene has been established on chromosome 2 (Zhao et al, 2003). As a result of only one other marker of Bt1 on chromosome 2 data exist, linkage maps cannot be obtained in this experiment. Besides, only the white RFLP and Bt2 are the markers that effect on chromosome 5.
A different set of molecular markers that are on chromosome 5 and chromosome 2 should be elected to map the wm gene and white RFLP more accurately. According to the Zhao et al. 2003, among other chromosomes, they have indicated Bt32, Bt4 and Bt14 microsatellites on chromosome 2. Moreover, a linkage map could be built up by examining chromosome 5 and the entire markers.
To sum up, the more significant number of offspring would also be favoured in this experiment. However, a smaller sampling size can be utilized to set up the linkage since the crossing over is not taking place in Bactrocera tryoni (Zhao et al., 2003).
- Frommer, M. and Bennett, C.L. (1997). The white gene of the tephritid fly Bactrocera tryoni is characterized by a long untranslated 5' leader and a 12 kb first intron. Insect Mol Biol. 6(4): 343-356.
- Griffiths, A.J.F., Wessler, S.R., Lewontin, R.C., Gelbart, W.M., Suzuki, D.T. and Miller, F.H. (2005). An Introduction to Genetic Analysis. 8th ed. pp. 128.
- Griffiths, A.J.F., Wessler, S.R., Lewontin, R.C. and Carroll, S.B. (2008). Introduction to Genetic Analysis. 9th ed. New York: Freeman. pp 146-149.
- The University of Sydney (2019). GEGE2001 Semester 2, 2019 – Genetics and Genomic Practical Manual. Pp 2,6-10 & 12.
- Zhao, J.T., Frommer, M., Sved, J.A. and Zacharopoulou, A. (1998). Mitotic and polytene chromosome analyses in the Queensland Fruit Fly, Bactrocera tryoni (Diptera: Tephritidae). Genome. 41(4): 510-526
- Zhao, J.T., Frommer, M., Sved, J.A. and Gillies, C.B. (2003). Genetic and Molecular Markers of the Queensland Fruit Fly, Bactrocera tryoni. J Hered. 94(5): 416-420