Abstract: Background and Objective: Tegal duck is a domestic waterfowl species known as a major producer of eggs that is traditionally used for making the famous salted egg by the local people. This study aimed to analyze the phylogenetic relationship of the Tegal duck through the gene encoding 18S ribosomal RNA (rRNA) and mitochondrial cytochrome oxidase I (COI). Methodology: We amplified and sequenced part of the 18S rRNA and COI gene of Tegal duck. These data were used to determine their similarity with other Anas species retrieved from GenBank and analyzing phylogenetic relationship. Results: The size of the PCR product of partial 18S rRNA was approximately 485 bp, whereas the size of the partial mitochondrial DNA (mtDNA) of the COI gene sequence was 751 bp. The 18S rRNA data showed that the Tegal duck is closely related to A. platyrhynchos (84.4% similarity). The COI gene data of the Tegal duck confirmed this relationship, with 99% homology with A. platyrhynchos voucher NHMO-BC399. However, the COI gene fragment of the Tegal duck also showed 99% homology with A. poecilorhyncha. Conclusion: Tegal duck has a close phylogenetic relationship with not only A. platyrhynchos but also A. poecilorhyncha.
INTRODUCTION
Duck is a dominant livestock with economic importance in Tegal, Central Java, Indonesia, which has the highest population after Brebes and Klaten Regencies. This region is popularly known as center for the highest quality salted duck eggs. Their egg production is unstable and showed tendency to decrease1. Morphological and physiological characterization has been commonly used for duck breeding programs. It resulted in instability of the progeny and the product2,3. The characteristics of Tegal ducks have been considered to be typologically similar with those of mallard (Anas platyrhynchos)2-4. Their variative phenotypes such as beak color, head color and tail, have been confusing in making decisions regarding in animal breeding4,5. Moreover, the origin of the domestic duck in Asia is still debatable6-8. Genetic characterization of this duck is important to improve egg production in Tegal. It is known that genetic variation is responsible for phenotypic traits. Therefore, it is very important to analyze genetic characteristics of the domestic Tegal duck for breeding program, with the possibility through their advantages. Genetic identification of the Tegal duck is conducted using two DNA barcoding methods consisting of nuclear and `mitochondrial DNA (mtDNA). The combination of mitochondrial and nuclear genes produce more credible species delineations than those based on a single gene9. Detection of nuclear DNA is limited due to the general low copy number of sequences and differs from the several copies of mtDNA per cell10,11. Mutation and recombination in the nucleus create variety in the nuclear genes, while mutation is the primary source of sequence variation in animal mitochondria. All processes can be the material for the identification of genetically related species10,11. Based on recent studies, COI gene has been effective to identify species in poultry because of their high mutation rate, which was sufficient to discriminate amongst species11. The advantage of mtDNA is supported at faster nucleotide substitution and lack of recombination11,12-14. In addition, mitochondrial genome is maternally inherited8,15-18. Thus, COI gene can be used in differentiating taxonomial class starting from the level of intraspecies to ordo, for example Copepoda evolution12,14. Another DNA barcodes suitable for animal identification is 18S rRNA gene, the central component of ribosome. This region is one of the most conserved genes in all cells14,19,20. Phylogenetic analysis based on molecular characterization of Tegal duck species will be important for searching a stable product for improving breeding strategies for Indonesian domestic ducks. Molecular identification of most members of the genus Anas has generally been performed using the COI mitochondrial gene. However, since 18S rRNA is also a reliable molecular barcode, it would be better if the phylogenetic analysis is performed using both of these genetic markers. In the present study, variations in mtDNA and ribosomal DNA is investigated to determine the phylogenetic relationships of the Tegal duck species at the molecular level.
MATERIALS AND METHODS
Specimen collection and DNA extraction: Tegal ducks at 3 months of age were obtained from the Center of Breeding Poultry in Banyubiru Semarang, Department of Animal Husbandry and Animal Health in Central Java Province, Indonesia. The DNA samples were extracted from the tissue samples of the Tegal ducks21.
PCR amplification and DNA sequencing: The amplification of the 18S rRNA and COI gene in the mtDNA was used extracted genomic DNA. The COI forward primer was 5-TTCTCCAACCACAAAGACATTGGCAC-3 and the reverse primer was 5-ACGTGGGAGATAATTCCAAATCCTG-3 22. For the 18S rRNA gene, a universal primer was used with the forward primer 5-GTAGTCATATGCTTGTCT-3 and the reverse primer 5-GCTGGCACCACACTTGCCCT-3. The PCR for the COI gene was performed in a Mini Thermal Cycler (Eppendorf, Germany). The initial denaturation step was conducted at 95°C for 5 min. The next steps were 35 cycles at 95°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec and a final extension step at 72°C for 7 min. The PCR for the 18S rRNA gene was used an initial denaturation step at 94°C for 2 min. The PCR consist of 30 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min. The final extension step at 72°C for 2 min. Purification and sequencing of the PCR products were performed by 1st Base Singapore.
Data analysis: For data analysis, published sequences of some members from the genus Anas were downloaded from GenBank. Sequence data of the duck COI gene and 18S rRNA are listed in Table 1. Sequences were aligned using the ClustalX program23. Identification of nucleotide variations in duck species and construction of the neighbor-joining phylogenetic tree were conducted using MEGA software version 5 (Japan)24. The phylogenetic tree was constructed by substitution model using the Jukes-Cantor model. It assumes equal base frequencies and equal mutation rates.
| Table 1: | The 18S rRNA and mitochondrial COI gene sequences of Anas species from GenBank used in this study |
The rate of nucleotide substitution is the same for every pair of nucleotides. Frequencies of A, T, C and G will eventually become equal to 0.2525,26.
RESULTS AND DISCUSSION
Amplification of 18S rRNA gene by universal primer: Figure 1 shows the products of the Tegal duck 18S rRNA amplification. The annealing temperature that showed positive bands and exhibited the best sequencing results was 55°C. These data confirmed that the primer we chose in this study was suitable for amplifying the conserved region in the Tegal duck. The size of the PCR product of the 18S rRNA fragment was approximately 485 bp.
| Fig. 1: | The 18S rRNA fragment of the Tegal duck |
| lane 1 = 18S rRNA fragment, lane 2 = 1 kb DNA ladder | |
These sequences showed high homology with 18S rRNA partial sequences homology with those of A. platyrhynchos (Fig. 2). Sequence of the 18S rRNA fragment with gene resources from GenBank showed approximately 99% similarity between the Tegal duck and A. platyrhynchos (accession number EU123318.1) from Saudi Arabia, A. platyrhynchos (accession number AF173614.1) from the USA and A. platyrhynchos (accession number D38362.1) from Ibaraki, Japan. Approximately 98% similarity was also observed between the Tegal duck and the A. platyrhynchos isolate Moulard AF23 (accession number FJ416869.1) but only in the short region (data not shown). The 18S rRNA sequences from Tegal duck and A. platyrhynchos species identified 98 and 99% similarity in the region. A. platyrhynchos was grouped with A. poecilorhyncha and had 0.0047% difference26. This result also supported the idea that the region with high intra-species conservation, such as the 18S rRNA genes with similarities close to 100%, could be used for species-level analyses in the case of copepods12. Analysis and comparison of the sequences of the Tegal duck and A. platyrhynchos identified six single nucleotide polymorphisms and one deletion in the conserved 18S rRNA region. The transition and transversion mutation can be seen in Fig. 2 accomplished by a deletion at position 474 bp.
The sequencing result of the PCR product followed by similarity analysis for the Tegal duck showed close homology with those of the 18S rRNA gene of A. platyrhynchos. The 18S rRNA sequences of the Tegal duck and A. platyrhynchos exhibited conserved regions between the sequences.
| Fig. 2: | Comparison of 18S rRNA sequences of the Tegal duck and the interspecies A. platyrynchos |
| Orange box indicates a transition mutation and blue box indicates a transversion mutation | |
The 18S rRNA region was also highly conserved of A. platyrhynchos (accession number EU123318.1) and A. platyrhynchos (accession number AF173614.1).
The sequencing results of the PCR product of the partial fragment of 18S rRNA gene of the Tegal duck showed close among species and had been used for species-level analyses for example A. rubripes8 and copepods12.
Amplification of Tegal duck COI gene: The Tegal duck’s partial fragment of the COI gene of approximately 751 bp are shown in Fig. 3. This COI gene fragment showed 99% similarity with sequences from A. poecilorhyncha and A. platyrhynchos.
Homology analysis revealed that the Tegal duck shared a common ancestor with A. poecilorhyncha and A. platyrhynchos, as illustrated on its cladogram (Fig. 4). Similarity analysis of the COI gene fragment of 751 bp length revealed that the Tegal duck, compared with some A. platyrhynchos species, had several base substitutions as detailed in Table 2. The difference between A. platyrhynchos and A. poecilorhyncha was the position of the COI gene in the complete mitochondrial genome. The A. platyrhynchos COI gene consistently stayed at position 6476-7225, whereas the A. poecilorhyncha COI gene was found at several positions, such as 5426-6175 in A. poecilorhyncha (accession number KF156760.1) and A. poecilorhyncha (accession number KC466567.1) but at other positions such as 6476-7225 in A. poecilorhyncha (accession number KF751616.1).
The cladogram on Fig. 4 indicates that some A. platyrhynchos and A. poecilorhyncha species had been bred and had a descendant possessing both of their characters, whereas others still retained the dominant original character of A. poecilorhyncha. The COI gene of Tegal duck showed a unique character combination resemble with 13 subspecies A. platyrhynchos and it also gained the character of 3 subspecies of A. poecilorhyncha. This result strongly implied that the domestic Tegal ducks from Indonesia may have ancestral A. platyrhynchos predominantly and also little character of A. poecilorhyncha species. Similarity analysis of the COI gene fragment with a length of 751 bp showed that the Tegal duck had several base substitutions almost in the same position as that in some A. platyrhynchos and A. poecilorhyncha species. We have resumed some differences in bases substitution, transition and transversion among A. platyrhynchos and A. poecilorhyncha as depicted in Table 2.
Further analysis also found 99-100% similarity in the COI gene of the Tegal duck with other members of the Anas genus as shown in Fig. 5. They exhibited some variations in this region, all with respect to species in the genus Anas. In all the COI gene fragments, the rate of transition was higher than the rate of transversion.
Similarity analysis of the COI gene sequences of the Tegal duck in comparison with the mitochondrial COI gene of other members of the Anas genus identified 99-100% similarity in their COI regions. However, the differences among members in the genus Anas is much greater than the difference between Tegal duck with A. platyrhynchos and A. poecilorhyncha.
Similarity analysis also revealed the number of base substitutions between the Tegal duck and A. platyrhynchos with transition mutation in three base positions and a transversion mutation in three base positions, which can be used to distinguish between species. This result creates a bias in the ratio of the number of transitions to the number of transversions. As shown in Table 3, the maximum likelihood estimation of transition/transversion bias (R) for all the sequences is 15.30, which was still within the range for mtDNA27.
| Fig. 3: | The COI gene fragment of the Tegal duck |
| lane 1 = 100 bp DNA Ladder, 2 = COI gene fragment | |
| Table 2: | Base substitutions in the Tegal duck COI gene fragment compared with those of several A. platyrhynchos and A. poecilorhyncha species |
| Fig. 4: | The phylogenetic relationship of the COI gene fragment of Tegal duck with A. platyrhynchos and A. poecilorhyncha |
| Table 3: | Maximum likelihood estimation of substitution matrix |
| The probability of substitution (r) from one base (row) to another base (column), *Rates of different transversion substitutions are shown, **Those of transition substitutions are shown | |
The probability of transition was greater than that of transversion, indicating that the changes in the COI gene of the Tegal duck could be silent mutations and therefore do not significantly alter the genes to ensure its taxon in the Anas group.
The amino acid analysis of the COI gene from the members of the Anas genus showed the characteristics of the mitochondrial genetic code, in which codon AUA is used to encode methionine. Furthermore, codon UGA, usually a termination codon, codes for tryptophan. The nucleotide frequencies are A = 24.06%, T/U = 22.38%, C = 36.39% and G = 17.17%. These COI regions generally have the highest transition/transversion ratios and are more significant in closely related species. In general, the percentage of amino acids in the COI gene fragment in Tegal duck is dominated by alanine (14.5%), followed by isoleucine (13.9%), leucine and glycine (13.2%). It is also observed that codons ending in C are always the most frequently observed in the COI gene of Anas, followed by codons ending in T and then A or G. Codons ending in C appear to be somewhat more frequently used than those ending in T, among the twofold degenerate codons. The least common third position nucleotide in all categories, except for arginine and glycine codons, is codon G (G having the same frequency with C and T but less than A). The number of base substitutions in the Tegal duck and A. platyrhynchos with both transition and transversion mutations in three base positions can be used to distinguish between species. The probability of transition substitution was higher than that of transversion substitution, wherein the substitution of adenine with guanine and vice versa was the greatest. This is affected by the similarity of their molecular structure. Therefore, the probability is smaller for transversion substitution, which is caused by the purine and pyrimidine nucleotides that have a different larger molecular structure27. The substitution at the nitrogen bases causes a mismatch in the sequence and there is turnover among the same base pairs (transitions) and the base pairs with its opponent (transversion).
The principal problem of evolution in the nuclear and mitochondrial genomes is a mutation that changes the genes. A gene or DNA sequence with a mutation will cause nucleotide substitution that might be spread into the population by natural selection and genetic drift and sometimes will be fixed in a species.
| Fig. 5: | Similarity analysis of the COI gene fragment of the Tegal duck compared with other members of the genus Anas |
| Fig. 6: | Phylogenetic evolutionary tree of the COI gene displaying the evolutionary relationship of the Tegal duck within a lineage shared by other members of Anas species |
| The scale bar in the upper right represents 0.05 substitutions per nucleotide site | |
This character will be inherited into their descendant species if the mutated gene produces new morphological or physiological characters. Single nucleotides can also cause polymorphisms in RNA that will exhibit genetic differences between individuals and have an important function when the mutation occurs in a specific region. A mutation in the 18S rRNA is a heritable change in the DNA sequence that may or may not potentially affect the phenotype of the organism, since the mutations are very important in producing genetic variations. These genes can influence the characteristics of an organism. Changes in any part of the environment could make the organism extinct. The theory of evolution assumes that mutations are random because of their adaptive value. For example, some nucleotides are more mutable than others. The COI regions generally have high transition/transversion ratios of mutation and are more significant in closely related species. Nucleotide substitution with a transition bias is a common phenomenon in animal mtDNA. Purified selection can aid in tolerating synonymous mutations and eliminating nonsynonymous mutations27. Transition mutations occur at a higher frequency than that of transversion mutations. Transitions are less likely to result in amino acid substitutions and are therefore more likely to be considered silent substitutions in populations28.
Phylogenetic analysis: Tree construction for a closely related gene using DNA sequences would be better than using protein because it avoids any silent substitution that is phylogenetically informative. The results of the phylogenetic analysis of the members of the Anas genus in Fig. 6 showed that the domestic Tegal duck from Indonesia could more likely to be a hybrid of A. platyrhynchos and A. poecilorhyncha as they are found together in a monophyletic cluster in their phylogenetic tree. Other duck species are well separated using the COI gene in mitochondrial DNA. This result was also supported by an adequate bootstrap value of approximately 94% (the number of bootstrap replications was 1000). The result shown in Fig. 6 also reveals that in addition to A. platyrhynchos and A. poecilorhyncha, the Tegal duck was also a member of the monophyletic cluster with A. laysanensis, A. rubripes, A. fulvigula and A. superciliosa. This result supports the finding that A. platyrhynchos species have hybridized extensively with other closely related species worldwide such as A. superciliosa in Australia and New Zealand, A. rubripes and A. platyrhynchos from North America29 and A. laysanensis30.
The genus Anas is primarily derived from mallard ducks. Moreover, the evolution of Anas species is a dominant factor that affects the production of hybrid ducks29-32. Based on the COI gene fragment of the Tegal duck analysis results which showed 100% homology with A. platyrhynchos voucher NHMO-BC399 (GenBank accession number GU571240), Scandinavian birds from the trans-Atlantic species33 which indicated that the Tegal duck originated from migrating wild ducks.
The results for the 18S rRNA gene of the Tegal duck confirmed by the results of the COI gene showed that it was a hybrid of A. platyrhynchos and A. poecilorhyncha with dominant character of A. platyrhynchos. Combination of both genetic marker had resulted more accurate molecular identification of species.
The results of the phylogenetic evolutionary tree analysis of the COI gene also support the phylogenetic analysis result using 18S rRNA, which showed that the Tegal duck have a common ancestor A. platyrhynchos and A. poecilorhyncha. This result is in agreement with another study which grouping A. platyrhynchos with A. poecilorhyncha. A phylogenetic relationship was shown among the Korean duck derived from the mallard (A. platyrhynchos) and the spot-billed duck (A. poecilorhyncha)34. However, another study reported that based on their mtDNA, the domestic Korean ducks originated only from A. platyrhynchos6. Some South and East Asian ducks may live in areas with suitable food and habitat because they are migratory birds and have characteristics by which they can be easily adapted26. Hybrids from mallards and spot-billed ducks often appear in natural habitats, which makes it difficult to identify. In current study, phylogenetic analysis also supported the result that the mallard and spot-billed duck breeds are not separated.
The COI regions generally have the highest transition/transversion ratios and are more significant in closely related species. These patterns are usually similar for almost all vertebrate groups, except Xenopus31. The Tegal ducks are genetically close to A. platyrhynchos as they show a closer relationship with other A. platyrhynchos species compared with A. poecilorhyncha. Moreover, the pairwise distance among the Tegal duck and other members of Anas demonstrated a relatively low-level interspecific distance. This smaller difference is possibly due to the crossbreeding of spot-billed ducks and mallards influenced by the same habitats32. Phylogenetic analysis also revealed that mallards and spot-billed ducks are genetically very close. The phylogenetic tree analysis also showed that in the monophyletic cluster, the Tegal duck has a close relationship with A. laysanensis from Hawaii, A. rubripes and A. fulvigula from Canada and A. superciliosa from Australia. These vast areas have been proven as the migratory habitats of most mallards, covering an area of several thousand kilometers. Mallard populations are highly genetically connected, which accounts for the decrease in the domestic mallard numbers and the genetic diversity exacerbated by alterations in the local environment6. Morphologically, mallard and spot-billed duck breeds are highly similar in shape, behavior and habitat and are even genetically compatible for crossbreeding32,33,35. Although animal mitochondrial sequences are known to evolve rapidly, their gene arrangements often remain unchanged over long periods of evolutionary time32,36-37.
The COI gene provided a better coverage and wider spectrum of species characterization than the 18S rRNA gene. The use of 18S rRNA in combination with the COI gene had overcome the limited availability of the data set in GenBank to conduct a more accurate analysis, which made it possible to find more genetic diversity within A. platyrhynchos. This study provides important information for improving egg production of Tegal duck. Since the egg production of A. platyrhynchos (average 140-150 each year) was higher than A. poecilorhyncha (84-108 each year)38-40, this study will help in developing the breeding program strategy and selection for the Tegal duck. Thus, a new approach on selection for identification and selection on superior domestic ducks based on the integration of morphological and physiological characterization with molecular characterization may be achieved.
CONCLUSION
In conclusion, phylogenetic analysis based on the nuclear 18S rRNA gene and mitochondrial COI gene characterization was gain representative identification of the Tegal duck. The Tegal duck had showed close genetic relationship with A. platyrhynchos and A. poecilorhyncha.
SIGNIFICANCE STATEMENTS
This study discover that integration of both genetic markers 18S rRNA and COI gene had provided a comprehensive genetic information for Tegal duck and showed their close similarities with not only A. platyrhynchos but also A. poecilorhyncha. The identification of domestic duck species and genetic variation would be very useful to gain consistency of the progeny and the egg production. Thus, this study provide a new approach on identification and selection on superior domestic ducks based on the integration of morphological and physiological characterization with molecular characterization to guide the development of breeding program for Tegal duck and possibly for another local duck in Indonesia.
ACKNOWLEDGMENTS
This research was part of program funded by Research Grant of Directorate General of Higher Education, The Ministry of National Education of Indonesia according to The letter of agreement on the implementation of Community Services Grant No.: 013/O06/PP/SP/2011 date 04 April 2011 which is gratefully acknowledged. We also thanks to The Center of Breeding Poultry on Banyubiru Semarang, Department of Animal Husbandry and Animal Health on Central Java Province, Indonesia, for providing the Tegal Duck to be used in this research.