Links between White RFLP and WM Phenotypes in B Tyroni

Introduction

Genetic markers, such as microsatellites, SNPs and RFLPs are differences in the nucleotide structure of DNA that allow for variation in the phenotype of an individual, however, this variation is not one that is visible. These genetic markers may be amplified and cut for examination by the use of PCR and a restriction enzyme digest (Vignal et al., 2002). Gel electrophoresis is utilised to amplify and separate the DNA of the organism that is being studied, allowing for easy viewing and comparison to known markers (Vignal et al., 2002). These genetic markers are thus able to be utilised to form a linkage map, allowing for an understanding of the linkage of differing genes at various loci in the chromosome (Vignal et al., 2002) (Gilchrist et al., 2014)

Bactrocera tryoni, otherwise known as the Queensland Fruit Fly or Q Fly is a species of tephritid that is commonly found on Australia’s east coast. B. tryoni is a destructive pest that can infest various commercial fruits that are grown along the east coast (Gilchrist et al., 2014). This is a large economic problem to Australia and has resulted in many tests being conducted to attempt to reduce the damage caused by this pest. The use of genetic and molecular markers in these experiments is essential as they provide us with the ability to examine and alter the genome of the fly species (Gilchrist et al., 2014). The sterile insect technique is a solid attempt at a solution for this problem. This technique involves breeding captive B. tyroni to select for sterility. A large population of these sterile flies are then released into a wild population, whereby they breed, eventually passing on their sterile genes to their offspring and destroying the wild fly population (Raphael et al., 2014).

This experiment was conducted in an attempt to determine whether there was any link between the white RFLP and WM phenotypes in B. tyroni, and through utilising this along with various microsatellite loci, attempt to map the white gene of this species to a chromosome.

Materials and Methods

A male backcross was performed, crossing an F1 male with phenotype +/WM Ra/Rb with a female whose phenotype is WM/WM Rb/Rb (The same as the phenotype of the mother of the F1) to produce a G2 progeny that may be tested.

18 μl of PCR master mix (made up of 67 mM Tris-HCl (pH 8), 16.6 mM [NH4]SO4, 0.45% triton X-100, 0.2 mg/ml gelatin, 3 mM MgCl2, 0.5mM nucleotide, dATP, dTTP, dCTP and dGTP), 2 μl of 1.087 μM White 2 and White 5’ extension primer mix and 3 μl of DNA template were added for each sample. A control was set up with the same ingredients listed above, however MilliQ water was substituted in place of the DNA template, then vortexed for 5 seconds before 2 μl of 0.7 units/ μl Taq polymerase was added. Samples were then placed in the cycler for 1 cycle at 95C for 3 minutes, followed by 35 cycles at 95C for 1 minute to denature. This was followed by 35 cycles at 60C for 1 minute to anneal, before being cycled 35 times at 72C for 1 minute and finally cycled once more at 72C for 5 minutes. The samples were then stored for a week at -20C.

The PCR product was placed in the centrifuge and spun for 2 seconds. 10 μl were then removed and placed in a sample. 6 μl of milliQ water, 2 μl of 10x buffer and 2 μl of 1.75 U/ μl of Rsal was added. Incubate for 1 hour at 37 C.

2 μl of 10x loading dye were added to control samples and reaction samples. Load 20 μl of pUC19/Hpall into the first well. Load 20 μl of 1kb ladder size standard into the last well. Load 12 μl of each control sample into a well of the agarose labelled ‘uncut’. Load 20 μl of each reaction sample into a well of the ‘cut’ agarose. Run the electrophoresis set up. Expose gels to UV transilluminator. Examine the gels to determine the phenotypes of your sample and use a chi squared test to determine the linkage of white RFLP and WM phenotypes to each other and to microsatellites located on different chromosomes.

Results

Multiple tests were conducted to determine the linkage between the WM visible marker and the white RFLP and various microsatellites present on differing chromosomes. The PCR that was conducted successfully displayed and amplified all but two samples that were loaded, one for each of the cut and uncut samples. This may have occurred as a result of insufficient or improper mixing of reagents when making up the PCR product. Results from Emily’s Wednesday group were taken to ensure accurate and reliable data could be used to fill in the missing wells. The uncut PCR plate was used as a control for the experiment. The G2 fragment sizes present on the PCR plate were found to be accurate as they were approximately the same as the parental fragments produced.

The first linkage analysis that was performed was done in order to determine whether there was linkage present between the white RFLP and WM visible makers in the G2 progeny of B. tryoni that was tested. This test demonstrated that there was roughly a 1:1:1:1 ratio present between the white RFLP and WM phenotype, as can be observed in Table 1. This indicates that there is no linkage between the white RFLP and WM phenotype

Table 1: A linkage analysis of the presence of the wm visible marker and white RFLP in B. tryoni

Phenotype of G2	Expected Ratio if Unlinked	Expected Ratio if Linked	Actual Observed Ratio
wm/wm Rb/Rb	1	1	3
+/wm Ra/Rb	1	0	4
wm/wm Ra/Rb	1	0	5
+/wm Rb/Rb	1	1	4

A secondary linkage analysis was conducted to determine whether the WM phenotype and white RFLP were linked to the Bt1 microsatellite and thus also linked to chromosome 5. A chi squared test was conducted on the varying phenotypes present within the sample to determine their linkage. The test was conducted assuming that there was linkage between the WM visible marker and the microsatellite, as well as the white RFLP and the microsatellite. As is evident in Table 2, the linkage analysis produced a ratio of 1:0:0:1 when the presence of each allele was examined. The chi squared test produced an X^2 result of 0, with 3 degrees of freedom, which through the use of Microsoft Excel, was used to calculate a p-value of > 0.99. Thus the WM phenotype was found not to assort independently, concluding that it is linked to the microsatellite Bt1 and is found on chromosome 2. However, the white RFLP was found to assort independently on Bt1, thus it is not linked to the microsatellite and is not present on chromosome 2.

Table 2: Linkage analysis of the WM visible marker and white RFLP on the Bt1 microsatellite, located on chromosome 5

Phenotype	Observed ratio	Expected ratio
Ra/Rb WT SL (Father)	4	4
Rb/Rb WM SS (Mother)	3	4
Ra/Rb WT SS	0	0
Ra/Rb WM SL	0	0
Ra/Rb WM SS	5	4
Rb/Rb WT SS	0	0
Rb/Rb WT SL	4	4
Rb/Rb WM SL	0	0

A final linkage analysis was then performed to attempt to establish whether the white RFLP was linked to any microsatellite loci and to which chromosome it mapped to. The Bt2 loci was tested with the white RFLP through the use of a chi squared test to obtain the data present in Table 3. The white RFLP was found to be linked to the Bt2 microsatellite loci on Chromosome 5 (p = 0.97) as independent assortment was observed. This indicates that the white gene in B.tryoni is located on chromosome 5.

Table 3: A linkage analysis of the white RFLP and the Bt2 microsatellite, located on Chromosome 5

Phenotype	Observed ratio	Expected ratio
Rb/Rb SS	7	8
Rb/Rb SL	0	0
Ra/Rb SS	0	0
Ra/Rb SL	9	8

Discussion

Through analysis of the results, the WM phenotype was able to be mapped onto the 2nd chromosome, as it was found to be linked to the Bt1 microsatellite loci. Thus, as a result, we were able to retain the null hypothesis (see supplementary information for null hypothesis). The results gathered conformed to previous data from experiments conducted by Zhao et al (2003), whereby they utilised a technique known as fluorescent in situ hybridisation to map the genome (Zhao et al., 2003).

The white RFLP was similarly linked to a different microsatellite locus labelled Bt2 and was mapped on the 5th chromosome. As a result, the null hypothesis stated in the supplementary information was retained. Similarly, this result was further strengthened as it correlated with data gathered through the experiments of Zhao et al (2003) and their exploration of genetic and molecular markers in B. tryoni (Zhao et al., 2003).

There is no possible way to determine and map the location of the microsatellite loci on either chromosome 2 or 5 through the results gathered by utilising the white RFLP and WM phenotype. In order to be able to map these accurately and improve upon the results that were gathered, other experiments and procedures may be performed. Various other microsatellites may be tested with the white RFLP and WM phenotype to determine their linkage with these microsatellites (Zhao et al., 2003). This may then be compared with draft genomes of B. tryoni to validate any results that are gathered (Gilchrist et al., 2014). Similarly, other techniques may be used to identify the loci present within these chromosomes, such as the use of gene databases to serve as a foundation for a comparative study (Krosch et al., 2019).

In order to improve upon the experiment that was conducted, more PCR tests should be run to allow for more accurate comparisons and determination of gathered results. Faults in the data, such as those missing in well 5 and 24 of figure 1 may then be cross referenced with the other samples to determine whether an error has occurred. This faulty data may then be disregarded, and the experiment may be repeated to ensure accuracy.

Due to the data that was gathered, this experiment was able to be completed and the linkage between the white RFLP and WM phenotype were accurately tested. These linkage relationships were then further tested with various microsatellite loci present on differing chromosomes and were thus able to be mapped.

Supplementary Information

Table 4: Phenotypic analysis of a performed male backcross in relation to varying microsatellite loci and the presence of the white RFLP. Missing data was filled in using data from Emily’s Wednesday group.

Fly	Sex	Wm	Bt1 Chr 2	Bt2 Chr 5	Bt5 Chr 3	Bt7 Chr 2	Bt11 Chr 6	Bt15 Chr 6	Bt17 Chr 4	White RFLP
Parent	M	WT	LL	LL	SS	LL	SS	LL	LL	Ra/Rb
Parent	F	wm	SS	SS	LL	SS	LL	SS	SS	Rb/Rb
F1	M	WT	SL	SL	SL	SL	SL	SL	SL	Ra/Rb
G2-1	F	WM	SS	SL	SL	SS	LL	SS	SL	Ra/Rb
G2-2	F	WM	SS	SL	LL	SS	SL	SL	SS	Ra/Rb
G2-3	F	WM	SS	SS	SL	SS	SL	SL	SL	Rb/Rb
G2-4	F	WM	SS	SS	LL	SS	SL	SL	SS	Rb/Rb
G2-5	M	WM	SS	SL	SL	SS	SL	SL	SL	Ra/Rb
G2-6	M	WM	SS	SL	SL	SS	LL	SS	SS	Ra/Rb
G2-7	M	WM	SS	SS	SL	SS	LL	SS	SL	Rb/Rb
G2-8	M	WM	SS	SL	SL	SS	LL	SS	SS	Ra/Rb
G2-9	F	WT	SL	SL	SL	SL	SL	SL	SS	Ra/Rb
G2-10	F	WT	SL	SS	LL	SL	LL	SS	SL	Rb/Rb
G2-11	F	WT	SL	SL	SL	SL	LL	SS	SS	Ra/Rb
G2-12	F	WT	SL	SS	SL	SL	SL	SL	SL	Rb/Rb
G2-13	M	WT	SL	SS	SL	SL	SL	SL	SS	Rb/Rb
G2-14	M	WT	SL	SL	SL	SL	LL	SS	SL	Ra/Rb
G2-15	M	WT	SL	SL	LL	SL	SL	SL	SS	Ra/Rb
G2-16	M	WT	SL	SS	LL	SL	LL	SS	SL	Rb/Rb

Figure 1: B. tryoni DNA cut by a PCR reaction using the Rsal enzyme.

Figure 2: Uncut DNA of B. tryoni amplified through the use of a PCR reaction.

Figure 3: Emily’s Wednesday group B. tryoni DNA cut by PCR reaction using Rsal (used to fill in missing data)

Table 2 Chi squared test:

Bt1

Chr2

Null = The white marks visible marker is linked to the Bt1 microsatellite on chromosome 2.

Alternate = The white marks visible marker is not linked to the Bt1 microsatellite on chromosome 2.

X^2 = sum of (O-E)^2/E

= (8-8)^2/8 + (0-0)^2/0 + (0-0)^2/0 + (8-8)^2/8

= 0

Degrees of freedom = 3

P value = >0.99

Therefore retain null hypothesis

Bt2

Chr 5

Assuming linkage

Null hypothesis: The white RFLP is linked to the Bt2 microsatellite on chromosome 5.

Alternate hypothesis: The white RFLP is not linked to the Bt2 microsatellite on chromosome 5.

X^2 = (O-E)^2/E

= (7-8)^2/8 + (0-0)^2/0 + (0-0)^2/0 + (9-8)^2/8

= 0.250

Degrees of freedom = 3

P value = 0.97

References

Gilchrist, A., Shearman, D., Frommer, M., Raphael, K., Deshpande, N., Wilkins, M., Sherwin, W. and Sved, J. (2014). The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species. BMC Genomics, 15(1), p.1153.

Krosch, M., Schutze, M., Strutt, F., Clarke, A. and Cameron, S. (2017). A transcriptome‐based analytical workflow for identifying loci for species diagnosis: a case study with Bactrocera fruit flies (Diptera: Tephritidae). Austral Entomology, 58(2), pp.395-408.

Raphael, Kathryn et al. (2014) Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique. BMC Genetics. [Online] 15 (Suppl 2), S9. [online]. Available from: http://search.proquest.com/docview/1629450779/.

Vignal, Alain et al. (2002) A review on SNP and other types of molecular markers and their use in animal genetics. Genetics, Selection, Evolution : GSE. [Online] 34 (3), 275–305.

Zhao, J., Frommer, M., Sved, J.A., C.B. Gillies., (2003). Genetic and Molecular Markers of the Queensland Fruit Fly, Bactrocera tryoni. Journal of Heredity, 94(5), pp.416-420.