Bactrocera tryoni is an extremely detrimental pest to citrus crops especially in Australia. At their peak, they have eliminated large proportions of citrus and stone fruits which is not only a loss in revenue to farmers but is also a health concern to consumers (H.V Weems, 2002). They have proven to be so devastating that they have attracted research on their lifecycle in an attempt to facilitate better pest control. However, this study explicitly expresses the need for more research, especially using more molecular markers, to eradicate Bactrocera tryoni (A.R Clarke, K.S Powell, C.W Weldon, 2010).
Molecular markers refer to the umbrella term describing various sequences of DNA which vary among individuals that are inherited without displaying any phenotypic differences (W.H Freeman, 2000). They can be used in tandem with visible markers to develop a genetic map of a species (E Coe, K Cone, M McMullen, 2002). This is achieved primarily by calculating recombination frequencies of the molecular markers to determine both the relative distance between genes and the order on which they lie on a chromosome (N.Sarawarthy, P.Ramalingam, 2011). Molecular markers have since overtaken visible markers in their effectiveness in constructing genetic maps as they are more effective in dealing with organisms such as flies that have phenotypes that are difficult to observe (F.Khan, 2015).
Genetic engineering studies have revealed that there are transformation mechanisms that aid in transferring genes to Bactrocera tryoni (M Koukidou, A Klinakis, C Reboulakis, 2006). In particular, a study has targeted gender determining genes to only yield male flies or to have females carry lethal genes which can be used to control population (K. Raphael, S.Whyard, D.Sheerman, 2004). This enlightens us to the potential to capitalise on with genetic mapping. We are therefore more motivated to seek solutions to problems such as pest control due to the advancements in genetic engineering techniques. This could prove to become a more sustainable method to control the population of Bactrocera tryoni in place of pesticides and insecticides. Such agents consequently result in the development of resistance of Bactrocera tryoni over time (L.J. Senior, B.P. Missenden, C.Wright, 2017).
In our experiment, we wanted build upon the genetic map of Bactrocera tryoni by testing if the visible white marks and the molecular white genes were linked. We did this by conducting a linkage analysis on the progeny from a male backcross of Bactrocera tryoni. We chose to do a male backcross as the male chromosomes do not cross over during meiosis which makes it very useful in determining if genes are linked. The aim of the experiment being to determine if the white marks and white genes are on the same chromosome. Additionally, the white and white marks genes could also be mapped onto specific chromosomes through the linkage analysis of 7 microsatellite DNA sequences.
Materials and methods
We looked at DNA from G2 progeny resulting from the male backcross. The F1 male used in the backcross came from a pure breeding father homozygous for the wild-type white marks and Ra alleles. Conversely, a pure breeding mother homozygous for the recessive white marks and Rb alleles.
We first amplified our DNA samples from the G2 progeny via Polymerase chain reaction (PCR) amplification of the white gene fragments. The essential materials needed included a thermal cycler and a centrifuge. The PCR protocol consisted of 18µl of the PCR master mix, 2µl of the Primer mix and 3µl of the DNA template itself. We then used 2µl of Thermostable DNA (Taq) polymerase mix at a concentration of 0.35 units per µl. It is important to note that the PCR protocol was made while the thin-walled tube was on ice to ensure that the Taq polymerase remained inactive until the PCR mixture was finally put into the thermocycler as this prevents non-specific amplification of DNA. This is to avoid DNA being amplified before the primers have had time to anneal to encapsulate the gene of interest.
We also set up a control where in one PCR protocol, the DNA sample is replaced with sterile milli-Q water.
After that we commenced our restriction enzyme digest of the PCR product. The essential materials we required was a water bath at 37℃, centrifuges, vortex and the restriction enzyme, RsaI. We used the restriction enzyme, RsaI (2.0 µl at a concentration of 1.75U/µl). 37℃ of incubation is used as it is roughly the optimum temperature for RsaI to work in. We also set aside half of the DNA sample as uncut DNA, to act as another control for the experiment. This is to set a reference in the event that if there were any experimental error with our PCR or restriction digest which caused abnormal DNA bands were produced during the gel electrophoresis, we would be able to detect it.
The final phase was when we conducted our gel electrophoresis, utilising a UV filter and a UV transilluminator both to visualise the movement of our cut and uncut samples.
We accomplished the aim of our experiment and concluded that the white and white marks genes are unlinked and are located on separate chromosomes. This is because the genotypes of our G2 progeny could only be possible if the genes were unlinked. Our results concluded that there are two RFLP phenotypes for the white gene when digested by RsaI, heterozygous at the R loci and homozygous at the R loci for the Rb allele from observing 4 and 2 DNA bands respectively. The length of the fragments were also similar to their predicted lengths, further confirming that the bands present are the bands resulting from the white gene (GEGE2 x 01, 2018). We calculated the fragment sizes using a standard curve has a strong correlation coefficient of 0.964, giving us greater confidence in the validity of our calculations.
We statistically tested our results, by performing a Chi-squared test with our null hypothesis being the scenario where the genes are unlinked. We calculated a large p-value between 0.90 and 0.95 which allows us to accept our null hypothesis. Hence, we can further conclude that the visible white marks marker and molecular white marker are unlinked and any differences in the offspring frequency between the four genotypes was largely due to chance.
Lastly we mapped out the location of the genes using microsatellite DNA. The white marks gene is found on chromosome 2 as it was inherited microsatellites Bt1 and Bt7 which are both found on chromosome 2. The white gene is found on chromosome 5 as it was inherited with microsatellite Bt2 found on chromosome 5. Altogether, we have achieved the aim of the experiment and mapped out where the white marks and white genes are located within the genome.
If our experiment looked at more genes of Bactrocera tryoni, we can eventually construct a genomic map for the species. To do this more easily, we could use other types of molecular markers such as amplified restriction fragment length polymorphisms (AFLPs). This is because one disadvantage of RFLP analysis alone is that we need to have prior insight on the sequences of a particular gene (A Varma, N N Shrivastava, 2017). Another example could be random amplification of polymorphic DNA (RAPD) where there is no requirement for specific primers (Q Yi, S Yi, T Qin, 2018). We could use a large DNA sample size to improve the validity of our results as our experiment only studied DNA from 16 offspring. Collectively, we can work towards mapping the entire genome of Bactrocera tryoni which researchers are close to doing, having already produced the draft genome for the species (A.S. Gilchrist, D Sheerman, M Frommer, 2014).
The genetic mapping of Bactrocera tryoni could have implications on population control in conjunction with CRISPR/Cas mediated gene editing. This is because CRISPR/Cas mediated gene editing requires specific sequences which a genetic map can provide (M Crossley, 2018). Hence, a stronger genetic map can help us to target genes to control the population of Bactrocera tryoni by causing flightlessness for example (W Zheng, Q Li, H Sun, 2018). Additionally, the combination of CRISPR/Cas mediated gene editing and better genetic maps allows us to know which genes can be used as gene insertion markers (A Handler, 2001). For example in our experiment, by mapping the white and white marks genes, we know that we can not use them as gene insertion markers as they are unlinked (B Miki, S Mchugh, 2004).
In conclusion, our experiment successfully determined that the white and white marks genes are not found on the same chromosome and that their loci are on chromosomes 2 and 5 respectively. The results are significant in terms of improving our ability to control the population of Bactrocera tryoni. This is either directly by altering critical genes for survival of Bactrocera such as flightlessness or indirectly by having a greater understanding of potential gene insertion markers such as the experiment we conducted. In both cases, there is the utilization of current CRISPR/Cas mediated gene editing.