Determination of sex is one of the key difficulties encountered in genetics. Considerable headway in the sex determination of mammals and fruit flies has been made in recent years. However, little has been done concerning the avian species and much of the available information concerns the domestic chicken or Gallus gallus. The availability of molecular markers makes it convenient to determine the sex of an organism using genetic techniques. In the past, the lack of appropriate molecular markers necessitated the utilization of morphological features such as the tarsometatarsi bone (Sadler 1991). Currently, universal molecular markers are available for determining the sex of many avian species. These markers are created by targeting well-preserved primer flanking regions within the chromo-helicase-DNA binding gene 1 (CHD1), present on birds’ sex chromosomes (Itoh et al. 2001). Ascertaining the sex of birds is imperative for confined breeding of threatened bird species as well as in fundamental research for instance developmental biology and molecular ecology (Itoh et al. 2001).
Female mammals have two similar chromosomes (XX) and are homogametic. Male mammals, on the other hand, have two different chromosomes (XY) and are said to be heterogametic. However, in birds, the females are heterogametic possessing the ZW chromosomes, whereas the males are homogametic possessing the ZZ chromosomes (Correa et al. 2005).
Sometimes it is necessary to determine the sex of a living bird species, a process that requires extraction of DNA from the bird. Three main methods are used to obtain DNA. These include invasive methods, destructive methods, and non-invasive methods. In destructive methods, the animal is killed before the sample is extracted for further investigation. Invasive techniques entail seizing the animal before drawing blood or extracting tissue. The use of polymerase chain reaction in the detection of genetic sexes of birds is useful since an extremely tiny sample, for example, a small blood droplet can yield an appreciable amount of DNA thereby minimizing trauma to birds (Itoh et al. 2001). Therefore, it is possible to obtain DNA samples from birds while avoiding destructive methods.
This practical aimed at extracting DNA from three tissue types (muscle, preserved blood and feathers), amplifying the CHD1 gene by PCR and carrying out gel electrophoresis to visualize the PCR products to establish the sex of the chicken.
Method
Extraction of DNA from Muscle Tissue
Qiagen DNeasy blood and tissue kit was used to purify DNA from chicken muscle. My tissue code was M13.48. A sterile blade was used to macerate 20mg of tissue into a pulp by chopping action in a petri dish. 180µl of ATL buffer was added to the tissue in a microcentrifuge tube and vortexed for approximately 15 seconds. Thereafter, 20µl of proteinase K was added, and the mixture was incubated at 56 oC for 30 minutes. 4µl of RNase was added to lyse the cells after which 200µl AL buffer and 200µl ethanol were thoroughly mixed with the sample to give a homogeneous solution. The sample was bound to a labeled collection tube by centrifuging at 8000 rpm for 1 minute. The flow-through liquid was disposed of, and the collection tube was replaced with a clean one (Hogan et al. 2013). 500µl of AW1 buffer was added and the sample centrifuged at 8000 rpm for 1 minute. This was repeated at 13000 rpm for 3 minutes. The remaining ethanol was taken out of the sample by centrifugation for 1 minute at the highest velocity. The extracted DNA was eluted into a clean 1.5ml microcentrifuge tube using 100µl of AE buffer after incubating for 1 minute followed by centrifugation for 1 minute at 8000 rpm. This yielded 200µl of DNA, which was stored at -20oC awaiting further analysis. The method of DNA extraction from other tissue types varied slightly. Full details are given in Hogan et al. (2013).
Electrophoresis
Agarose gel was prepared by boiling 1% agarose (w/v) in 1X TAE electrophoresis buffer in a microwave. The molten gel was allowed to cool to 50 oC in a water bath. GelRed stock solution was then added to the gel after which it was poured into the casting tray with the combs and allowed to set. The sample was prepared by mixing 10µl of genomic DNA with 2µl of 6X loading-dye in a clean microfuge tube. TAE buffer was poured over the gel after which 5µl of molecular markers λ Hind III and 2-log ladder were added into the initial and last wells (Hogan et al. 2013). The DNA sample was loaded into a blank well, and the well position was noted. The gel was run for 60 minutes at 120 volts, which separated the DNA according to size. The power was disconnected when the gel finished running after which the gel was visualized using Gel Doc system. The concentration of DNA was determined using a spectrophotometer using UV light at 260-280 nm.
Polymerase Chain Reaction
DNA was amplified using Taq polymerase by following the instructions in the provided kit. The master mix was prepared by pipetting each reagent in the appropriate order. The ready PCR tubes were positioned into the thermocycler and were run by “a program optimized to amplify the CHD1W and CHD1Z gene variants with the 2550F -2718R primer set” (Hogan et al. 2013, p.26). 10µL of the amplified PCR products were mixed with of 2µl of 6X loading dye and electrophoresed in 1% agarose gel in sodium borate buffer at 300V for 20 minutes. The amounts of the PCR outputs were then established by matching their positions with the standard bands in the marker.
Results
DNA Extraction
Figure 1: Gel of DNA extraction. There were six lanes in total. The first lane was the muscle tissue sample followed by the second muscle sample and the blood sample. The first lane had two bands although the upper band was not conspicuous. The second lane had no visible bands, whereas the third lane had one conspicuous band with red fluorescence. Lane 12 was the positive male control and had one conspicuous band. Lane 13 was the female positive control and had one conspicuous band (the bottom band) just like in lane 9. It was clearly seen that the negative controls did not have any band.
Table 1 shows the SDS gel obtained after the extraction of DNA from three tissue types as well as the apparent sex from the extracted DNA fragments before the amplification by PCR. Blood tissue yielded more DNA than muscle tissue. The sex of DNA samples in my group was female because of the two bands that were seen for each sample.
PCR of Extracted DNA
Figure 2: PCR Products for the three tissue types together with the positive and negative controls. Lanes 9 to 13 are muscle, muscle, and blood PCR products. The bands were thicker and more conspicuous than in the unamplified DNA. My sample had two clear bands, whereas only one band was visible before the amplification.
Table 1: Estimated concentration of the PCR products of the three tissue types
The sexes of the samples shown on the gel were all females apart from the positive control for the male sample. The approximate base pair size of the W gene was 0.5 kilobases, whereas the Z gene was about 3.0 kilobases. There were three female samples and no male sample. It was seen that the Z chromosome was substantially bigger in males than in females. The results obtained from the PCR correlated with the actual sex of the individuals.
The concentration of the DNA in ng/µl was calculated by dividing the mass of DNA in ng (obtained from the molecular weight marker) by the volume of sample DNA (in µl) that was loaded.
There were differences in the amplification between tissue types. Blood samples amplified more than muscle and feather samples. However, the negative controls did not amplify.
Discussion
There were differences in the concentrations of DNA obtained from different tissues. Blood samples contained the highest concentration of DNA followed by muscle tissue. Feathers contained the least DNA. Although my group did not have any feather samples, the content of DNA obtained from feathers was the least as observed from the gel images of groups with feather samples. The bands in my group were similar to those of a different group with muscle tissue, which had a faint upper band (Z chromosome).
The observed results were consistent with the expected results. According to Dubiec and Zagalska-Neubauer (2006), avian blood has nuclei with very large amounts of DNA compared to cells from feathers and muscle tissues, which do not have as many nuclei as blood hence the difference in quantities of DNA. Feathers can only yield high quality of DNA if they are freshly plucked. High quality DNA produces clear, bright and distinct bands, whereas DNA of poor quality yields faint bands.
There was no band visualized in lane 13. This was probably because the amount of DNA extracted was too little to be visualized or the sample was contaminated during DNA extraction. It was seen that the Z chromosome in males was significantly larger than the Z chromosome in females. This observation was consistent with previous studies done by Mendonça et al. (2010) and Chue et al. (2010), which suggested that the Z chromosome in males had more genes than the Z chromosome in females.
It was important to store samples properly before genetic analysis to maintain the integrity of the DNA. Incorrect storage of samples led to the degradation of DNA thereby producing low quality (low molecular weight) fragments. This was achieved refrigerating the samples. A few samples in the experiment had evidence of DNA degradation. For example, sample code M in lane 4 (bottom row) produced light, smeared bands implying that it was low quality or fragmented DNA.
Non-invasive procedures are the most secure and humane approaches for extracting DNA from animals. They are extremely useful when studying endangered species or animals that are hard to capture. Non-invasive methods do not interfere with the animal’s habitat and take advantage of substances left behind by the animal such as feathers and eggshells that contain DNA. Their shortcoming is that the DNA disintegrates with time hence making analysis difficult. In addition, non-invasive methods give low yields of DNA thereby necessitating advanced techniques of obtaining quality DNA for analysis.
Universal primers used in this experiment provided information about the sex of an individual. Most techniques that determine the sex of birds employ hybridization and magnification of primers related to the CHD gene. Such techniques have been used in the identification of over 50 bird species (Vucicevic et al. 2013). Universal primers locate sex-specific DNA, which is later amplified and visualized.
The advantages of employing universal sexing molecular markers include accuracy, cost, ease, and speed. Using primers that flank the CHD gene to identify the sex of birds is cheap, fast, accurate, and relatively easy to perform. The entire process involves extraction of DNA, PCR and resolution of the PCR products on gel. These steps can take as little as 5 hours (Dubiec and Zagalska-Neubauer 2006). In addition, there are three universal primers that can be used. These are 1237L/1272H P2/P8 and 2550F/2718R (Dubiec and Zagalska-Neubauer 2006). It is, however, imperative to check the available literature to establish the most reliable primers for a given species. In instances where such data is unobtainable P2/P8 and 2550F/2718R primers can be tried for their feasibility (Dubiec and Zagalska-Neubauer 2006).
The disadvantage of universal sexing molecular markers is that different primers use different ways of assigning sex. For example, the use of P2/P8 primers causes problems in the assignment of sex due to polymorphisms in the Z chromosome, which is documented in 20 species (Dubiec and Zagalska-Neubauer 2006). Therefore, sex is assigned based on the sizes of the fragments. Conversely, when using the 2550F/2718R primers sex is assigned based on the number of bands seen. All in all, it can be concluded that CHD-based sex identification is the most reliable known method of sex identification in birds.
References
Chue, J & Smith, C. A 2011, “Sex determination and sexual differentiation in the avian model,” FEBS Journal, vol.278 no. 2011, pp. 1027–1034.
Correa, S. M., Adkins-Regan, E., & Johnson, P. A 2005, “High progesterone during avian meiosis biases sex ratios toward females,” Biology Letters, vol.1 no.2005, pp. 215-218.
Dubiec, A., & Zagalska-Neubauer, M 2006, “Molecular techniques for sex identification in birds,” Biological Letters, vol.43 no.1, pp.3-12.
Hogan, F., Loke, S., & Sherman, C 2013, SLE254 Genetics practical manual 2013, Deakin University.
Itoh, Y., Suzuki, M., Ogawa, A., Munechika, I., Murata, A & Mizuno, S 2001, “Identification of the sex of a wide range of carinatae birds by PCR using primer sets selected from chicken EE0.6 and its related sequences,” The American Genetic Association, vol.92 no.2001, pp. 315-321.
Mendonça, M. A. C., Carvalho, C. R., & Clarindo, W. R 2010, “DNA content differences between male and female chicken (Gallus gallus domesticus) nuclei and Z and W chromosomes resolved by image cytometry,” Journal of Histochemistry & Cytochemistry, vol 58 no.3, pp. 229–235.
Sadler, P 1991, “The use of tarsometatarsi in sexing and aging domestic fowls (Gallus gallus L.), and recognizing five-toed breeds in archaeological material,” Circaea, vol.1 no.8, pp. 41-48.
Vucicevic, M., Stevanov-Pavlovic, M., Stevanovic, J., Bosnjak, J., Gajic, B., Aleksic, N., & Stanimirovic, Z 2013, “Sex determination in 58 bird species and evaluation of CHD gene as a universal molecular marker in bird sexing,” Zoo Biology, vol.32 no.3, pp. 269-273.