Morphology and Genetic Diversity of Mitochondrial Dna D-loop Region Using Pcr-rflp Analysis in Magelang Duck and Other Native Duck

The aim of this study was to investigate the different of plumage colors on morphological diversityof Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelangduck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck) in Indonesia. Bloodsample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristicsof body measurement, production ability and egg quality of Magelang duck were analyzed usingCompletely Randomized Design with 11 plumage colors as treatments. PCR technique was administeredto amplify fragments in mtDNA D-loop region and PCR products were digested with endonucleaserestriction enzyme AluI and HaeIII. The result showed that morphology diversity of Magelang duck wasstatistically affected by different plumage colors. PCR-RFLP analysis using AluI and HaeIII restrictionenzyme resulted in six combinations of restriction fragment pattern shown in six haplotypes (A, B, C, D,E and F). Haplotype difference showed genetic diversity in the population of Magelang duck and theother native ducks. In conclusion, the different plumage colors affected morphology diversity ofMagelang duck. Genetic diversity of Indonesian native duck population could be identified by usingPCR-RFLP analysis on mtDNA D-loop region.


INTRODUCTION
Magelang duck is native duck thriving in Muntilan, Magelang, Central Java province with specific and ultimate morphological characteristics of relatively big body, high egg production and various plumage color. Ismoyowati and Purwantini (2010) reported that Magelang duck qualitatively has the highest plumage color diversity compared to other native duck.
Individual genetic variation within population is the figure of the heritable genetic expression variation. Genetic expression is the characteristic possessed by an individual as distinctive morphological feature. Through DNA marker diversity analysis on protein or enzyme, individual genetic diversity within and between population can be identified (Sakai et al., 1998). One of the analysis methods to identify genetic in several fowl species applies Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism (PCR-RFLP) (Herman, 2004;Sartika, 2007). Mitochondrion DNA lies outside the nucleus in one cell compartment or organelle named mitochondrion (Zhao et al., 2004), with higher mutation rate (5-10 times) than DNA nucleus (Parsons et al., 1997;Brown et al., 1979;Sigurδardóttir et al., 2000in Sudoyo, 2004, so it has high discrimination ability (Marzuki, 2004). Likewise, there are one closed circular area with complete necluotide sequence and one non coding region called displacement loop (D-loop) measuring 1049 pb in duck (GenBank: HM010684.1, 2010). Tsai et al. (2009) proved the complete D-loop mtDNA structure in Columba livia that can be used to identify the mother and genetic linkage.
PCR-RFLP technique is a technique that can multiply certain DNA fragment to figure out whether or not restriction site difference exists in DNA fragment inter individual within one family or population (Griffiiths et al., 2003), which enables to develop as alternative method to analyze genetic diversity and individual genetic uniformity and diversity in native duck population. PCR-RFLP analysis has been well applied to differ species and to detect interspecies or intraspecies variation in some animals like tuna, lobster and swine (Wolf and Hubner, 1999), that has served as verification tool of food product from various animal (Lockley and Bardsley, 2000;Rojas et al., 2011), and is used as well to identify meat characteristic on peacock (Pavocristatus) and other fowl like chicken and turkey (Saini et al., 2007). A mtDNA PCR-RFLP method also identifies genetic diversity in population of wild quail and laboratory quail (Shen et al., 1999), and other fowl species as chicken (Gallus gallus), turkey (Meleagris gallopavo), duck (Anas platyrhynchos) and goose (Anser anser) (Wisniewska and Slota, 2009;Silva et al., 2009).
Research on the effect of plumage color difference in Magelang duck morphology has never been done. In addition, information of identification and molecular genetic diversity using mtDNA D-loop region PCR-RFLP analysis of native ducks in all over Indonesia is limited as well. This research aimed to figure out the influence of different plumage colors on morphological diversity of Magelang duck and genetic diversity based on PCR-RFLP mtDNA Dloop region analysis of Magelang duck and other native duck populations in Indonesia.

MATERIALS AND METHODS
Materials used to study morphology diversity were 50 six-month-old Magelang ducks at early production with 11 different plumage colors, namely A. Jarakan polos (plain brown), B. Bosokan (dark brown), C. Klawu blorok (light brown and white), D. Kalung ombo (brown with wide white collar), E. Kalung ciut (brown with thin white collar), F. Cemani (plain black), G. Gambiran (dark brown and white), H. Jarakan kalung (brown with white collar), I. Jowo polos (brown with dark brown spot), J. Wiroko (black and white), K. Plain white (yellow bill and feet). Each color consisted of five ducks, except for A and B, each had 3 and 2 ducks, respectively.
Blood sample was taken from 50 Magelang ducks and 20 were from each native local ducks (Tegal, Mojosari, Bali and Alabio ducks). Chemical reagent used to extract DNA was DNA Isolation Kit (Geneaid). PCR-RFLP analysis used KAPA (Kit PCR), primer Forward (DL-AnasPF), primer Reverse (DL-AnasPR), dH 2 O free nuclease, endonuclease restriction enzyme AluI and HaeIII (Thermo Scientific product, Lithuania) and enzyme buffer. Chemicals for agarose gel were agarose powder, buffer 0.5x TBE, good view, loading day and DNA ladder.
The experiment was conducted to figure out (1) morphology diversity and (2) genetic diversity. Morphological diversity was analyzed using Completely Randomized Design (Steel and Torrie, 1980). The treatments were 11 different plumage color with 2-5 ducks replicates. The observed variables were (1) egg production, (2) egg weight (g/egg), (3) egg quality comprising egg length and width, egg index, eggshell thickness, albumen and eggyolk weight, albumen height, Haugh Unit (HU) and yolk color, (4) body weight (g), (5) body length (cm), (6) breast perimeter (cm) and (7) neck length (cm). Body parts measurement was done three times to avoid empirical mistakes of measurement. Egg production and weight was recorded three times and egg quality was randomly observed, in which 10 samples represented each plumage color. Data were analysed with analysis of variance.
Mathematical method used was: Y ij = µ + αi + εij Where Y ij = result of observed variables µ = mean of population αi = influence of plumage i th number εij= error Egg production measured was hen day production (HDP), thus the number of egg according to one month of individual record was divided by the number of female ducks times 100% (Ensminger, 1992), while egg weight was the whole weight of the egg weighed on digital scale with 0.01 g accuracy. Egg length was the length from the pointed end and obtuse end of egg, while egg width was the distant of 2 sides of egg in the centre, measured with digital slide compass with 0.01 mm accuracy. Egg index was the comparison of egg width and length times 100% (Reddy et al,. 1979), while eggshell thickness was measured with digital slide scale from three angles: the pointed end, center and obtuse end of egg, then the score were averaged. Albumen and yolk were separated using egg separator then each weight was scaled and the height was measured with Haugh Unit (HU) (Raymond Haugh, 1937in Monira et al., 2003. Formula used was: HU = 100 x log (h -1.7 w 0.37 + 7.6). Where HU = Haugh Unit h = albumen height (in milimeter) w = egg weight (in gram) Yolk color was observed by comparing it to Roche yolk colour fan.
Genetic diversity was analyzed by extracting DNA from duck blood sample and amplifying mtDNA D-loop region with PCR technique and the analysis applied PCR-RFLP. Blood sample was taken using disposible syringe from vena axillaries, then collected in vacuntainer containing EDTA. Total genome DNA was extracted using Isolation Kit Geneaid according to the protocol. DNA extract was used as PCR template without purification process and resulted in reproducible PCR products. Amplification process of mtDNA D-loop region was conducted with primer DL-AnasPF (L56) 5' -GTTGCGGGGTTATTTGGTTA-3' and DL-AnasPR (H773) 5'-CCATATACGCCAACCGTCTC-3', applying GeneAmp R PCR system thermocycler 2400 (Perkin Elmer). PCR reagent solution comprised 12.5 µl KAPA (Kit PCR), 1 µL primer Forward (DL-AnasPF) 10 pmol, 1 µL primer Reverse (DL-AnasPR) 10 pmol, 9.5 µL dH 2 O free nuclease and 1 µL DNA template. PCR cycle consisted of predenaturation at 94 o C for 5 minutes, denatuation at 94 o C for 30 seconds, annealing at 55 o C for 45 seconds, elongation (extention) at 72 o C for 1 minutes and post elongation at 72 o C for 5 minutes. PCR reaction was repeated 35 times for optimum result. PCR products were separated by electrophoresis in 1.5% low-melting agarose gel using buffer 0.5x TBE in Submarine Electrophoresis (Hoefer, USA). RFLP analysis administered PCR products digested with endonuclease restriction enzyme AluI and HaeIII. As much as 1 µL endonuclease restriction enzyme added with buffer R 2 µL and 7 µL dH 2 O free nuclease was collected in PCR microtube, then added with 5 µL PCR product. The solution was then incubated at 37 o C overnight for 16 hours. PCR-RFLP analysis result that has been cut with restriction enzyme and undergone electrophoresis in 2% agar gel then was observed for the DNA band resulted to know the digestion success. DNA band figure in agar gel was taken using digital camera, tabulated then analyzed for the genetic diversity. DNA band obtained from the visualization of agar gel electrophoresis on each sample was then used to determine the haplotype, genetic uniformity and diversity inter Magelang duck and other native ducks. Haplotype diversity obtained showed genetic diversity in Magelang duck and the other native ducks.

Morphology Diversity in Magelang Duck Population
This research well demonstrated that plumage color affected (P>0.05) morphology diversity comprising morphological   (Table 1), egg production (Table 2) and egg quality (Table 3) in Magelang duck population. Many genes determined plumage color pattern and interacted with other genes to determine the phenotype; however, information on the location of gene that controls plumage in specific chromosome was still limited and mechanism underlying this pattern was not absolute either (Stevens, 1991). Color formation of animal's plumage, eyes and skin was affected by melanin pigment and the synthesis is catalyzed by tyrosinase enzyme (Price andBontrager, 2001 andLiang et al., 2010). Single locus, melanocortin-1 reseptor (MC1R), is responsible to melanic polymorphism. MC1R has various roles among different species (Mundy, 2005). Hormone that limits plumage color expression in most birds is estrogen-dependent found in the females. Mutation point in MC1R and TYRP1 was figured to be responsible to produce pigmentation variants, and TYRP1 expression is lower in the female than in the male (Irwin, 1994). The more frequent plumage color change of the female is mostly due to dichromatism , assuming that sexual selection is more likely to happen to the female's plumage color (Burns, 1998).
This research showed that egg production and its weight was lower but the eggshell thickness was higher (Table 2) than the result of Ismoyowati and Purwantini (2010), reporting that egg production and egg weight of Magelang duck were 70.24±14.10% and 69.19±4.05 g, respectively, and the eggshell thickness was 0.38±0.02 mm. According to Sofwah (2007), eggshell quality was affected by egg weight and mother's age. This production diversity was influenced by the different of laying period, cage environment, and feed given.