Mapping Genes to Chromosomes in Bactrocera Tryoni

Introduction

The Queensland fruit fly, Bactrocera tryoni, is considered Australia's most economically damaging tephritid pest due to its infestation of almost all fruit crops. Traditional pest control methods involving insecticides have been ecologically adverse, prompting the exploration of genetic population desiccation methods like the Sterile Insect Technique (SIT) as an alternative (Langford et al., 2014).

In the case of the Mediterranean fruit fly, Ceratitis capitata, the SIT relies on genomic maps to control reproduction by utilizing Y-autosome translocations, which link males to specific alleles, allowing the release of only infertile males (Zhao et al.

, 2003). The mapping of the B. tryoni genome holds the potential to implement similar mechanisms for safeguarding affected crops.

Furthermore, the striking morphological similarities between B. tryoni and related species, such as B. neohumeralis, make it difficult to distinguish them at the larval stage. However, genome mapping may unveil diagnostic genomic variations that can assist in quarantine testing and hybridization efforts (Gilchrist et al., 2014).

Molecular markers are DNA polymorphisms that lack observable phenotypic effects and can be analyzed as alternatives to visible markers (Griffiths et al.

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, 2000). These markers encompass repeated sequences of transallelically varying lengths, known as microsatellites, and polymorphisms at restriction enzyme binding sites, leading to the prevention of enzyme binding and cleavage, known as Restriction Fragment Length Polymorphisms (RFLP). In the case of B. tryoni, an RFLP is associated with the white gene, which does not exhibit any observable phenotype, and microsatellites have been mapped to each chromosome. A recessive mutant visible marker, known as white marks (wm), expresses white thoracic marks, while wild types display a yellow phenotype (GEGE2X01, 2019).

Genome maps of Anopheles Gambiae and Aedes aegypti have previously been constructed using microsatellite and RFLP analysis (Langford et al., 2014).

In male B. tryoni, meiotic recombination is generally absent, resulting in the paternal co-inheritance of genes on the same chromosome (complete linkage). However, genes located on different chromosomes assort independently (Zhao et al., 2003). Linkage analysis involving dihybrid male and double homozygous recessive female crosses may thus reveal co-chromosomal loci.

This experiment aims to map the white and white marks genes onto the B. tryoni genome by utilizing the white RFLP and microsatellite loci.

Materials and Methods

In this section, we describe the materials and methods employed in our study to map the white and white marks genes in Bactrocera tryoni.

B. tryoni exons 4B contain PCR primer White 2 and white 5' ext annealing sites, flanking a 680bp DNA fragment containing two RsaI restriction sites (GTAC), one of which (RsaI*) is polymorphic for adenosine. We amplified this region using Polymerase Chain Reaction (PCR) and subsequently digested it with RsaI. The Ra allele recognizes both RFLP sites with the GTAC sequence, resulting in the production of 360, 190, and 130 bp DNA fragments upon digestion. In contrast, the Rb allele carries a single nucleotide polymorphism (SNP) in RsaI*, leading to the generation of 550 and 130 bp fragments. The digested products were analyzed using gel electrophoresis. Additionally, we recorded 7 microsatellite genotypes.

Unlinked genes will sort independently and will produce both recombinant and parental phenotype progeny. Linked genes will be inherited together, resulting in no phenotypically non-parental offspring, and the phenotypic ratio will be 1:1 of the parental phenotypes. Similarly, all gene combinations will exhibit 1:1 ratios of parental phenotypes if linked, and all parental and non-parental phenotypes if unlinked.

The PCR reaction mixture consisted of PCR master mix (67 mM Tris-HCl, pH 8; 16.6 mM [NH4]SO4; 0.45% Triton X-100; 0.2mg/ml gelatin, 3 mM MgCl2, 0.5mM nucleotide (dATP, dTTP, dCTP, dGTP)), 12.5µM white 2 and white 5’ext primer mix, and 0.35 units/µl Taq-polymerase, all kept on ice. The components were added in the following order to a 0.2mm thin-walled tube on ice: PCR master mix, primer mix, and DNA template. The mixture was briefly spun for 5 seconds, followed by the addition of Taq polymerase. As a control, a negative reaction without DNA was conducted. The reaction tubes were subjected to a thermal cycler for approximately 3 hours.

The samples were stored at -20°C for a week.

Half of the PCR product was digested with the restriction enzyme RsaI ("cut"), while the remaining half was left uncut as a control. A solution comprising 10% (v/v) 10X restriction buffer, 50% (v/v) PCR product (from previous steps and pre-prepared controls), 10% (v/v) and 0.175 units/µL RsaI enzyme was prepared sequentially in MQ water. The digest solution was spun and incubated in a 37°C water bath for 60 minutes.

The PCR products were stained with bromophenol blue and subsequently run on agarose gels (2% agarose, 10% v/v SYBR safe, sodium borate buffer (36mM Boric Acid, 10mM NaOH)) at 180V for 30 minutes, alongside a pUC19/Hpall DNA and 1kb ladder standard.

The gels were visualized under a UV transilluminator.

Results

Initially, the cut gels failed to exhibit the expected band patterns. Consequently, we conducted another electrophoresis analysis on successfully digested DNA to determine the results.

Our findings indicate that the white and white marks genes in Bactrocera tryoni are located on separate chromosomes.

Interestingly, some experimental progeny displayed non-parental phenotypes, contrary to the expectations of gene linkage. We performed a chi-squared test under the null hypothesis that the white and white marks genes assort independently. The resulting p-value exceeded 0.9, leading to the conclusion that the null hypothesis cannot be rejected.

In Table 1, we present the results of our linkage analysis of white marks and white RFLP genes. Notably, there are non-zero results for non-parental genotype progeny, indicating independent linkage. Here, "Wt" indicates the wild-type phenotype, and "wm" indicates the white marks phenotype.

Considering that there is a lack of recombination in male B. tryoni, independent assortment must be attributed to independently assorted chromosomes, rather than genetically distal genes, as even distant genes are inherited together.

Our analysis of the White RFLP gene showed a linkage to chromosome 5.

Table 2 displays the results of our linkage analysis of the White RFLP gene and a microsatellite on chromosome 5. Notably, there are no non-parental phenotype progeny.

The Bt2 microsatellite marker, located on chromosome 5 (GEGE2X01, 2019), exhibited complete linkage to the White RFLP gene. All Rb homozygotes were homozygous for the short repeated sequence allele (S), while RFLP heterozygotes were all heterozygous for the repeated sequence alleles. The Ra/Ra male parent P1 expressed homozygosity for the long allele (L). These results strongly suggest that the Ra allele is inherited with L, and the Rb allele is inherited with S. Consequently, the White RFLP and Bt2 markers coexist on chromosome 5. A chi-squared test confirmed this linkage with a p-value exceeding 0.95.

The White Marks gene displayed linkage to chromosome 2.

Table 3 illustrates the results of our linkage analysis of the White Marks gene and microsatellites Bt1 and Bt7. As expected, there are no non-parental phenotype progeny, and a 1:1 ratio of parental progeny is observed. Here, "SS" represents homozygosity for the short repeated sequence allele, "SL" represents heterozygosity for the microsatellites, "wm" denotes the white marks phenotype, and "wt" stands for the wild-type phenotype.

These results unequivocally confirm the complete linkage of the White Marks gene to the Bt1 and Bt7 microsatellite markers on chromosome 2. All individuals with the mutant white marks phenotype were homozygous for the short allele, while wild-type progeny, heterozygous for wm/wm+, were all heterozygous for both microsatellites. The wild-type P1 exhibited homozygosity for the long allele. A chi-squared test yielded a p-value exceeding 0.99, providing strong support for this conclusion.

Discussion

Our results are consistent with the findings of Zhao et al. (2003), who successfully located the white gene on autosome 5 and the white marks gene on autosome 2, utilizing microsatellite linkage analysis.

The uncut gel electrophoresis results exhibited a singular band ranging between 500-750bp, aligning with the expected size of the 680bp PCR products. The absence of unexpected band patterns serves as confirmation that the digested DNA was not contaminated with differently sized DNA fragments, affirming that the PCR amplified the expected fragment length. In comparison to the cut results, this control experiment validates that the RsaI enzyme was the key factor responsible for digestion.

The absence of a band in the no-DNA negative control demonstrates that no foreign DNA contaminated the PCR or digestion processes, except for the steps where DNA was intentionally added to the PCR reaction or the restriction enzyme to the digest.

All of the cut digests, except for the pre-prepared P1, P2, and F1 controls, failed to produce the expected band patterns. The observed band sizes in the failed digests match those of the PCR products, suggesting that the RsaI sites were not cleaved. The discrepancy between the successful pre-prepared controls and the failed samples digested on the day of the experiment points to a potential issue in the methodology applied on that particular day. Possible causes for this failure include the inactivation of RsaI due to improper storage conditions between digesting the controls and the progeny samples, such as exposure to temperatures above -20°C, or suboptimal buffer conditions (Thermo Fisher Scientific, n.d.).

Although we successfully mapped genes to chromosomes through linkage analysis with molecular and visible markers, it is essential to acknowledge that our results do not provide information about the gene order and relative positions on the chromosomes. Genetic distance between loci can be estimated through recombination frequency analysis involving a dihybrid female crossed with a homozygous recessive male, as recombination predominantly occurs in females. Determining the physical order of genes may require a three-point test cross, although the orientation remains unknown without reference to known loci. It is important to note that these methods can be relatively inefficient and may not be suitable for distinguishing genetic distances greater than 50 centimorgans.

A contemporary approach to genome mapping involves whole-genome sequencing, as demonstrated by Gilchrist et al. (2014), who successfully assembled an estimated 93.6% of core B. tryoni genes. This methodology was also employed in sequencing the Ceratitis capitata genome, followed by annotation (Gilchrist et al., 2014).

However, it is important to acknowledge that the complete mapping of the B. tryoni genome remains a challenge due to repeat sequences that hinder current methodologies. Further innovation is required to address and sequence these repetitive regions to achieve a comprehensive genome map. Nonetheless, the progress made in mapping the genome is valuable, particularly in the context of rapidly developing and implementing the Sterile Insect Technique (SIT) for B. tryoni control (Dyck et al., 2005).

In conclusion, our study has successfully mapped genes of interest to chromosomes, aligning with existing literature through linkage analysis with both molecular and visible markers. Nevertheless, the specific gene order and relative positions on the chromosomes remain obscured, necessitating further analysis and the exploration of alternative methods for achieving a comprehensive understanding of the genome.

Supplementary Information

Here, we provide supplementary information related to the statistical tests conducted in our study.

Chi-squared Test of Progeny

For the final column, we applied the following formula:

Null Hypothesis: White and white marks are assorted independently

For the chi-squared test of progeny, we calculated a χ² value of 0.5 with degrees of freedom equal to 3. The resulting p-value falls within the range of 0.9 to 0.95, indicating that the null hypothesis, which suggests independent assortment of white and white marks, is not rejected.

Null Hypothesis: White RFLP and Chr 5 microsatellite Bt2 alleles are completely linked; Rb is linked to the short (S) allele and Ra to the long (L) allele.

For the chi-squared test concerning the linkage of White RFLP and Chr 5 Microsatellite Bt2 alleles, we calculated a χ² value of 0.25 with degrees of freedom equal to 3. The resulting p-value is in the range of 0.975 to 0.95, which supports the conclusion that the null hypothesis is not rejected.

Null Hypothesis: White marks and Bt1 and Bt7 microsatellites on Chr2 are completely linked. Wild type is linked to L, and white marks are linked to S.

For the chi-squared test regarding the linkage of White Marks and Bt1/Bt7 Microsatellites, a χ² value of 0 was calculated with degrees of freedom equal to 7. The resulting p-value exceeds 0.99, indicating that the null hypothesis is not rejected.

References

• Dyck, V., Hendrichs, J., & Robinson, A. (2005). Sterile insect technique. Dordrecht: Springer.
• GEGE2X01. (2019). Gene mapping with molecular markers. Sydney: University of Sydney, pp. 2-17. Available at: https://canvas.sydney.edu.au/courses/13041/pages/weeks-3-and-4-gene-mapping-with-molecular-markers?module_item_id=382236
• Gilchrist, A. S., Shearman, D. C. A., Frommer, M., Raphael, K. A., Deshpande, N. P., Wilkins, M. R., Sherwin, W. B., & Sved, J. A. (2014). The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species. BMC Genomics, 15.
• Griffiths, A., Wessler, S., Carroll, S., & Doebley, J. (2000). An Introduction to genetic analysis. 7th ed. New York: W. H. Freeman.
• Langford, E., Nielsen, U., Johnson, S., & Riegler, M. (2014). Susceptibility of Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), to entomopathogenic nematodes. Biological Control, 69, 34-39. Available at: https://www.researchgate.net/publication/259141756_Susceptibility_of_Queensland_fruit_fly_Bactrocera_tryoni_Froggatt_Diptera_Tephritidae_to_entomopathogenic_nematodes
• Thermo Fisher Scientific. (n.d.). 7 Common Issues with Restriction Digestion Reactions and How to Avoid Them. Thermo Fisher Scientific - UK. Available at: https://www.thermofisher.com/au/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/7-common-issues-with-restriction-digestion-reactions-and-how-to-avoid-them.html
• Zhao, J., Frommer, M., Sved, J., & Gillies, C. (2003). Genetic and Molecular Markers of the Queensland Fruit Fly, Bactrocera tryoni. Journal of Heredity, 94(5), 416-420. Available at: https://academic.oup.com/jhered/article/94/5/416/2187459