PCR-RFLP Analysis of D-Loop mtDNA in Indonesian Domestic Waterfowl

R Susanti, Fidia Fibriana, Ari Yuniastuti


Phylogenetic or population genetic studies have been focused on finding genetic variations in mitochondrial DNA (mtDNA) because mutations in this region contained five times higher than other genetic materials. The aim of this research was to generate PCR-RFLP D-loop mtDNA profiles of some Indonesian domestic waterfowl. The samples were 35 local ducks, 5 muscovy duck, and 5 geese. These local ducks included on seven types of Javanese ducks in Central Java, i.e. Magelang duck, Peking duck, Pengging duck, Tegal Branjangan duck, Tegal Jarakan duck, Tegal Blorong duck, and Tegal Lemahan duck. PCR technique was used to amplify the D-loop genes, and RFLP analysis of the D-loop was performed with AluIand RsaI. The results show that muscovy duck has no restriction sites in D-loop gene. Moreover, goose has a restriction site of AluI in D-loop gene at 550 bp and 187 bp; and it has no restriction site of RsaI. The analysis of RsaI-RFLP in mtDNA D-loop region of ducks resulted in 1 restriction fragment pattern which can be applied in the identification of duck to differentiate it from goose and muscovy duck. PCR-RFLP analysis duck D-loop mtDNA using AluI and RsaI restriction enzyme resulted in 3 combinations of restriction fragment pattern shown in 3 haplotypes (A, B, and C). Genetic diversity of Indonesian domestic waterfowl population could be identified by using PCR-RFLP analysis on mtDNA D-loop region. So far, there are few results of research on Indonesian domestic waterfowl genetic characteristics based on PCR-RFLP mtDNA D-loop marker. Also, this research has a good contribution on genetic characterization of local duck. The characterization is an important aspect to maintain breed quality and manage the genetic resources of Indonesian germplasm and in facilitating the breeding program.


Indonesian domestic waterfowl; mtDNA D-loop; PCR-RFLP

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Allendorf, F. W., & Luikart, G. (2009). Conservation and the genetics of populations. Hoboken: John Wiley & Sons.

Arif, I., & Khan, H. (2009). Molecular markers for biodiversity analysis of wildlife animals: a brief review. Animal Biodiversity and Conservation, 32(1), 9-17.

Atalan, E. (2001). Restriction Fragment Length Polymorphism Analysis (RFLP) of Some Streptomyces Strains from Soil. Turkish Journal of Biology, 25(4), 397-404.

Avise, J. C. (2012). Molecular markers, natural history and evolution: Springer Science & Business Media.

Bjrnstad, G., & Red, K. (2002). Evaluation of factors affecting individual assignment precision using microsatellite data from horse breeds and simulated breed crosses. Animal Genetics, 33(4), 264-270.

Brown, T. A. (2016). Gene cloning and DNA analysis: an introduction. Hoboken: John Wiley & Sons.

Calo-Mata, P., Sotelo, C. G., Prez-Martn, R. I., Rehbein, H., Hold, G. L., Russell, V. J., . . . Rosa, C. (2003). Identification of gadoid fish species using DNA-based techniques. European Food Research and Technology, 217(3), 259-264.

Dovc, P., Kavar, T., Slkner, H., & Achmann, R. (2006). Development of the Lipizzan horse breed. Reproduction in domestic animals, 41(4), 280-285.

Feare, C. J., & Yasu, M. (2006). Asymptomatic infection with highly pathogenic avian influenza H5N1 in wild birds: how sound is the evidence? Virology Journal, 3(1), 96.

Fibriana, F., & Hadiyanti, L. N. (2016). Phylogenetic Relationships of Local Durian Species based on Morphological Characteristics and PCR-RFLP Analysis of the Ribosomal Internal Transcribed Spacer (ITS) DNA. Biosaintifika: Journal of Biology & Biology Education, 8(3), 361-369.

Frankham, R. (2003). Genetics and conservation biology. Comptes Rendus Biologies, 326, 22-29.

Frankham, R., Briscoe, D. A., & Ballou, J. D. (2002). Introduction to conservation genetics: Cambridge University Press.

Glowatzki?Mullis, M., Muntwyler, J., Pfister, W., Marti, E., Rieder, S., Poncet, P., & Gaillard, C. (2006). Genetic diversity among horse populations with a special focus on the Franches?Montagnes breed. Animal Genetics, 37(1), 33-39.

Grunenwald, H. (2003). Optimization of polymerase chain reactions. PCR protocols, 89-99.

Hao, Y. (2009). Genetic diversity analysis of mitochondrial D-loop region of Cherry Valley duck breed. Journal of Anhui Agricultural Science, 134, 279-282.

Hold, G. L., Russell, V. J., Pryde, S. E., Rehbein, H., Quinteiro, J., Vidal, R., . . . Santos, A. T. (2001). Development of a DNA-based method aimed at identifying the fish species present in food products. Journal of agricultural and food chemistry, 49(3), 1175-1179.

Hu, J., Fang, S.-G., & Wan, Q.-H. (2006). Genetic diversity of Chinese water deer (Hydropotes inermis inermis): implications for conservation. Biochemical genetics, 44(3-4), 156-167.

Idaghdour, Y., Broderick, D., Korrida, A., & Chbel, F. (2004). Mitochondrial control region diversity of the houbara bustard Chlamydotis undulata complex and genetic structure along the Atlantic seaboard of North Africa. Molecular Ecology, 13(1), 43-54.

Iyengar, A., Diniz, F. M., Gilbert, T., Woodfine, T., Knowles, J., & Maclean, N. (2006). Structure and evolution of the mitochondrial control region in oryx. Molecular phylogenetics and evolution, 40(1), 305-314.

Leekaew, P., Songserm, T., Choothesa, A., & Boonyaprakob, U. (2008). A simple method to extract mitochondrial DNA in a non-invasive phylogenetic study of domestic native Thai ducks. Kasetsart Journal of Social Sciences, 42(42), 41-50.

Ludwig, A., Debus, L., & Jenneckens, I. (2002). A molecular approach to control the international trade in black caviar. International Review of Hydrobiology, 87, 661-673.

Mannen, H., Kohno, M., Nagata, Y., Tsuji, S., Bradley, D., Yeo, J., . . . Nomura, K. (2004). Independent mitochondrial origin and historical genetic differentiation in North Eastern Asian cattle. Molecular phylogenetics and evolution, 32(2), 539-544.

Matitaputty, P. R. (2012). Peningkatan produksi karkas dan kualitas daging itik melalui persilangan antara itik Cihateup dengan itik Alabio. Dissertations. Bogor Agricultural University.

Mirhendi, H., Makimura, K., Khoramizadeh, M., & Yamaguchi, H. (2006). A one-enzyme PCR-RFLP assay for identification of six medically important Candida species. Nippon Ishinkin Gakkai Zasshi, 47(3), 225-229.

Mller, A. P., & Erritze, J. (1998). Host immune defence and migration in birds. Evolutionary Ecology, 12(8), 945-953.

Moura, A. E., Tsingarska, E., D?browski, M. J., Czarnomska, S. D., J?drzejewska, B., & Pilot, M. (2014). Unregulated hunting and genetic recovery from a severe population decline: the cautionary case of Bulgarian wolves. Conservation Genetics, 15(2), 405-417.

Moyer, C. L., Tiedje, J. M., Dobbs, F. C., & Karl, D. M. (1996). A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. Applied and Environmental Microbiology, 62(7), 2501-2507.

Onuma, M., Suzuki, M., & Ohtaishi, N. (2006). Possible conservation units of the sun bear (Helarctos malayanus) in Sarawak based on variation of mtDNA control region. Japanese Journal of Veterinary Research, 54(2-3), 135-139.

Pedrosa, S., Uzun, M., Arranz, J.-J., Gutirrez-Gil, B., San Primitivo, F., & Bayn, Y. (2005). Evidence of three maternal lineages in Near Eastern sheep supporting multiple domestication events. Proceedings of the Royal Society of London B: Biological Sciences, 272(1577), 2211-2217.

Pfeiffer, I., Voelkel, I., & Brenig, B. (2005). Phylogenetics of the European Dahomey miniature cattle based on mitochondrial D?loop region DNA sequence. Animal Genetics, 36(2), 179-181.

Purwantini, D., Yuwanta, T., & Hartatik, T. (2013). Polymorphism of D-loop mitochondrial DNA region and phylogenetic in five Indonesian native duck population. International Journal of Poultry Science, 12(1), 55.

Rojas, M., Gonzlez, I., Pavn, M., Pegels, N., Hernndez, P., Garca, T., & Martn, R. (2011). Mitochondrial and nuclear markers for the authentication of partridge meat and the specific identification of red-legged partridge meat products by polymerase chain reaction. Poultry science, 90(1), 211-222.

Roux, K. H. (2009). Optimization and troubleshooting in PCR. Cold Spring Harbor Protocols, 2009(4), pdb. ip66.

Russell, V. J., Hold, G. L., Pryde, S. E., Rehbein, H., Quinteiro, J., Rey-Mendez, M., . . . Rosa, C. (2000). Use of restriction fragment length polymorphism to distinguish between salmon species. Journal of agricultural and food chemistry, 48(6), 2184-2188.

Saini, M., Das, D., Dhara, A., Swarup, D., Yadav, M., & Gupta, P. (2007). Characterisation of peacock (Pavo cristatus) mitochondrial 12S rRNA sequence and its use in differentiation from closely related poultry species. British poultry science, 48(2), 162-166.

Sotelo, C. G., Calo-Mata, P., Chapela, M. J., Prez-Martn, R. I., Rehbein, H., Hold, G. L., . . . Izquierdo, M. (2001). Identification of flatfish (Pleuronectiforme) species using DNA-based techniques. Journal of agricultural and food chemistry, 49(10), 4562-4569.

Sunnucks, P. (2000). Efficient genetic markers for population biology. Trends in ecology & evolution, 15(5), 199-203.

Toro, M., Barragan, C., & Ovilo, C. (2003). Estimation of genetic variability of the founder population in a conservation scheme using microsatellites. Animal Genetics, 34(3), 226-228.

Wang, C., Liang, Z., Yu, W., Feng, Y., Peng, X., Gong, Y., & Li, S. (2011). Polymorphism of the prolactin gene and its association with egg production traits in native Chinese ducks. South African Journal of Animal Science, 41(1).

Webster, R. G., Krauss, S., Hulse-Post, D., & Sturm-Ramirez, K. (2007). Evolution of influenza A viruses in wild birds. Journal of wildlife diseases, 43(3_Supplement), S1.

Wolf, C., Burgener, M., Hbner, P., & Lthy, J. (2000). PCR-RFLP analysis of mitochondrial DNA: differentiation of fish species. LWT-Food Science and Technology, 33(2), 144-150.

Wolf, C., Rentsch, J., & Hbner, P. (1999). PCR? RFLP analysis of mitochondrial DNA: A reliable method for species identification. Journal of agricultural and food chemistry, 47(4), 1350-1355.

Wu, H.-L., Wan, Q.-H., & Fang, S.-G. (2006). Population structure and gene flow among wild populations of the black muntjac (Muntiacus crinifrons) based on mitochondrial DNA control region sequences. Zoological science, 23(4), 333-340.

Xu, X., Wu, X., & Yu, Z. (2010). The mitogenome of Paphia euglypta (Bivalvia: Veneridae) and comparative mitogenomic analyses of three venerids. Genome, 53(12), 1041-1052.

Zhang, C., Wang, Y., Xie, M., Chen, M., Wang, X., & Hou, S. (2010). Mitochondrial coding gene polymorphisms associated with carcass traits in Beijing duck. Journal of Animal and Veterinary Advances, 9(19), 2522-2525.

Zhang, F., & Jiang, Z. (2006). Mitochondrial phylogeography and genetic diversity of Tibetan gazelle (Procapra picticaudata): implications for conservation. Molecular phylogenetics and evolution, 41(2), 313-321.

DOI: https://doi.org/10.15294/biosaintifika.v9i3.12177


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