Sex identification of different species of wild birds using a single universal protocol to the bird sexing method based on gene polymorphism
AbstractThis article presents an elaboration of the protocol for the method of sexing wild birds based on the polymorphism of the CHD gene using P2/P8 primer for Common Pheasant – Phasianus colchicus (Linnaeus, 1758; Galliformes, Phasianidae); Silver Lofur or Silver Pheasant – Lophura nycthemera (Linnaeus, 1758; Galliformes, Phasianidae), Budgerigar – Melopsittacus undulatus (Shaw, 1805; Psittaciformes, Psittacidae), Herring Gull – Larus argentatus (Pontoppidan, 1763; Charadriiformes, Laridae), and White Stork – Ciconia ciconia (Linnaeus, 1758; Ciconiiformes, Ciconiidae). Blood samples were taken from Common Pheasant, Silver Pheasant and White Stork using the “drop of blood on paper” method. For the Budgerigar and the Herring Gull, DNA was isolated from the feather follicle. To isolate DNA, a commercial NeoPrep 100 DNA reagent kit (Neogen, Ukraine) was used. Primers P2/P8 were used for PCR; PCR was performed using GenPac PCR Core reagents (Neogen, Ukraine). We selected the optimal amount of Tag polymerase, the amount of DNA and primers and, according to the amount of reagents, set acceptable amplification modes and electrophoresis agarose gel percentage. Prior to PCR, additional DNA gel electrophoresis purification is proposed, which increases the percentage of positive sex determination results. It was found that the ideal mixture for the 5 bird species was an amplification mixture (total volume 20 µL, containing 1 U Tag polymerase, 100 ng DNA and 0.6 µM of each primer). The amplified CHD-Z fragment of Common and Silver pheasants is of ~340 n. p., CHD-W ~360 n. p. Herring Gull and Budgerigar have ~350 n. p. of CHD-Z length, and ~400 n. p. of CHD-W length, White Stork has its CHD-Z of ~ 370 n. p. long. It is advisable to investigate the genome of the experimental bird species using horizontal electrophoresis in agar’s gel with the concentration of 5%, which makes it possible to clearly visualize the female genotype. The universal protocol of the method of sex determination based on polymorphism of the CHD gene for the 5 studied bird species is described. These results of the study led to the conclusion that for the simultaneous sexing of several species of birds, it is advisable to develop a unified protocol for determining the status of the CHD gene, with the aim of clarifying the gender, as well as new approaches in ornithology and ecology aimed at determining interspecific differences associated with gene polymorphism. Identification of differences in fragment sizes may be useful for identifying the species in cases when birds form mixed pairings for taxonomic and phylogenetic comparisons.
Bondarenko, Y., & Omar, H. (2013). Sravnitelnaya harakteristika i sovremennaya klassifikaciya metodov opredeleniya pola molodnyaka selskohozyajstvennoj pticy (analiticheskij obzor) [Comparative characteristics and modern classification of methods for determining the sex of young poultry (analytical review)]. Visnik Sumskogo Nacionalnogo Agrarnogo Universitetu, 23, 111–120 (in Russian).
Cerit, H., & Avanus, K. (2007a). Sex identification in avian species using DNA typing methods. World’s Poultry Science Journal, 63, 91–99.
Chaplygina, A., & Yuzyk, D. (2016). The analysis of heavy metal concentrations in eggs of collared flycatchers, Ficedula albicollis (Passeriformes, Muscicapidae), and tits, Parus major, Parus caeruleus (Passeriformes, Paridae), in different areas of North-Eastern Ukraine. Vestnik Zoologii, 50(3), 259–266.
Chaplygina, A., Pakhomov, O., & Brygadyrenko, V. (2019). Trophic links of the song thrush (Turdus philomelos) in transformed forest ecosystems of North-Eastern Ukraine. Biosystems Diversity. 27(1), 51–55.
Chaplygina, A., Pakhomov, O., Yevtushenko, H., & Brygadyrenko, V. (2020). Trophic links of the chaffinch (Fringilla coelebs) in transformed forest ecosystems of North-Eastern Ukraine. Biosystems Diversity, 28(1), 92–97.
Chaplygina, A., Savynska, N., & Brygadyrenko, V. (2018). Trophic Links of the spotted flycatcher, Muscicapa striata, in transformed forest ecosystems of North-Eastern Ukraine. Baltic Forestry, 24(2), 304–312.
Cheng, Y., Kuo, T., Lee, D., & Weng, C. (2006). Sex identification of the black-faced spoonbill (Platalea minor). Zoological Studies, 45(1), 104–113.
Dawson, D., Remedios, N., & Horsburgh, G. (2016). A new marker based on the avian spindlin gene that is able to sex most birds, including species problematic to sex with CHD markers. Zoo Biology, 35(6), 533–545.
Drahulian, M., Chaplygina, A., Savynska, N., Kostenko, S., & Buchek, P. (2018). Clinical blood analysis of the great tit Parus major. ScienceRise, 3, 11–14.
Drahulian, M., Chaplygina, A., Savynska, N., Kostenko, S., Ostrovsky, P., & Gusar, K. (2018). The physiological and genetic differences between flycatcher (Ficedula albicollis vs. Ficedula hypoleuca). Folia Oecologica, 45(2), 111–119.
Duan, W., & Fuerst, P. (2001). Isolation of a sex-linked DNA sequence in cranes. Journal of Heredity, 92, 392–397.
Dubiec, A., & Zagalska-Neubauer, M. (2006). Molecular techniques for sex identification in birds. Biological Letters, 43(1), 3–12.
Dybus, A., Siemierz, A., Wysocki, D., Szatkowska, I., Muszyńska, M., & Guenzel, S. (2009). Evaluation of the applicability of polymerase chain reaction (PCR) to sex identification in eurasian blackbirds (Turdus merula). Biological Letters, 46(1), 15 –20.
Ellegren, H. (2001). Hens, cocks and avian sex identification: A quest for genes on Z or W? EMBO Reports, 2(3), 192–196.
Fomin, E. (2008). Sery’j popugaj zhako [The African Grey Parrot ]. Veche, Moscow.
Griffiths, R., Double, M., Orr, K., & Dawson, R. (1998). ADNA test to sex most birds. Molecular Ecology, 7, 1071–1075.
Han, J., Jang, H., Cheong, S., Kim, S., Park, S., & Na, K. (2009). Sex determination by PCR-RFLP in the oriental white stork Ciconia boyciana. Zoological Studies, 48(5), 619–624.
He, X., Qing, B., Han, J., & Ding, C. (2013). Improved molecular assay for sex identification of the endangered crested ibis (Nipponia nippon) based on the CHD1 gene and a sex-linked microsatellite locus. Zoological Science, 30(9), 742–747.
Ito, H., Sudo-Yamaji, A., Abe, M., Murase, T., & Tsubota, T. (2003). Sex identification by alternative polymerase chain reaction method in Falconiformes. Zoological Science, 20, 339–344.
Kasuga, K., Higashi, M., Yamada, T., Sugiyama, T., Taniguchi, Y., Iwaisaki, H. (2012). The W- and Z-linked EEO.6 sequences used for molecular sexing of captive japanese crested ibis on Sado island. Animal Science Journal, 83(1), 83–87.
Kim, M., Lee, S., Lee, H., & Lee, S. (2012). Molecular tools for species and sex identification in the mixed-species flocks of bean geese and white-fronted geese. Zoological Science, 29(11), 761–765.
Kulibaba, R. (2015). Utvorennya geterodupleksnoyi DNK pri amplifikaciyi fragmentiv geniv TGF-b2 ta CHD u ptahiv [The approval of heteroduplex DNA with amplification of fragments of TGF-β2 and CHD genes in birds]. Scientific and Technical Bulletin of the Institute of Animal Husbandry of the National Academy of Agrarian Sciences of Ukraine, 114, 77–83 (in Ukrainian).
Kulibaba, R., & Ruda, S. (2012). Opredelenie pola strausov s ispolzovaniem polimeraznoj cepnoj reakcii [Determination of sex of ostriches using polymerase chain reaction]. Ptakhivnytstvo, 68, 256–262 (in Russian).
Mudrik, A., Kashentseva, T., Hamburg, E., & Gavrikova, E. (2013). Non-invasive method for identifying the gender of crane chicks by dna from capillary vessels of allantois. Ontogenes, 44(5), 372–376.
Nesterenko, O. (2014). Geneticheskie metody opredeleniya pola: Problemy i sposoby ih resheniya [Genetic methods for determining sex: Problems and methods for solving them]. Scientific Research in Zoological Parks, 30, 63–84 (in Russian).
Ogawa, A., Solovei, I., Hutchison, N., Saitoh, Y., Ikeda, J., Macgregor, H., & Mizuno, S. (1997). Molecular characterization and cytological mapping of a non-repetitive DNA sequence region from the Wchromosome of chicken and itsuse as a universal probe for sexing Carinatae birds. Chromosome Research, 5, 93–101.
Reddy, A., Prakash, V., & Shivaji, S. (2007). A rapid, non-invasive, PCR-based method for identification of sex of the endangered Old World vultures (white-backed and long-billed vultures) – Implications for captive breeding programmes. Current Science, 92(5), 659–662.
Robertson, B., & Gemmell, N. (2006). PCR-based sexing in conservation biology: Wrong answer from an accurate methodology? Conservation Genetics, 7, 267–271.
Sacchi, P., Soglia, D., Maione, S., Meneguz, G., Campora, M., & Rasero, R. (2004). A non-invasive test for sex identification in short-toed Eagle (Circaetus gallicus). Molecular and Cellular Probes, 18(3), 193–196.
Serebryakov, A. (2010). Perepela: Soderzhanie, kormlenie, razvedenie [Quail: Maintenance, feeding, breeding]. Penza Press, Penza (in Russian).
Vuchichevich, M. (2014). Analiza CHD gena ptica kao molekularnog markera za determinaciјu pola [Analysis of the CHD gene in birds as a molecular marker for sex determination]. Belgrade (in Serbian).
Wang, N., & Zhang, Z. (2009). The novel primers for sex identification in the brown eared-pheasant and their application to other species. Molecular Ecology Resources, 9, 186–188.
Wang, Z., Zhou, X., Lin, Q., Fang, W., & Chen, X. (2011). New primers for sex identification in the Chinese egret and other ardeid species. Molecular Ecology Resources, 11(1), 176–179.
Wysocki, D. (2006). Factors affecting the between-season divorce rate in the urban population of European blackbird Turdus merula in North-Western Poland. Acta Ornithologica, 41, 71–78.
Zhang, Р., Han, J., Liu, Q., Zhang, J., & Zhang, X. (2012). Sex identification of four penguin species using locus-specific PCR. Zoo Biology, 32, 257–261.
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