Indian Journal of Animal Genetics and Breeding 26 (1,2): 26-30, January - December 2008 Genetic divergence between Red Jungle fowl and other domesticated poultry species using 12S rRNA gene polymorphism JATYASHI GUPTA, ANUP SINGH, R R CHURCHIL, B P SINGH and DEEPAK SHARMA Genome Mapping Laboratory, Central Avian Research Institut, Featnagar, Utar Pradesh 243 122 ABSTRACT ‘A~450 bp fragment of 12S RNA gene was amplified by using universal primers in Red Jungle fowl (RUF) and sequenced. Nucleotide sequence comparisons for 12S tRNA sequences from different chicken breeds / poultry species showed ample variation among them. Sequence alignment of partial 12S rRNA ‘gene showed that per cent genetic similarity between RIF and poultry species ranged from 68.9 to 98.2, hile genetic divergence ranged from 0.7 % ~ 15 %. Red jungle fowl showed maximum genetic similarity ‘with chicken (99.2%), while least genetic similarity with duck (77.4 %). Among other comparisons, except the lower genetic similarity of duck with all other poultry species (> 78 %), the genetic similarity of almost similar magnitude (82.8 % to 86.3 %) was observed. Key words : Phyogenetic relationship, 12S rRNA gene polymorphism, Red Jungle fowl Evolution can be conceived as a three step process - evolution and speciation of wild ancestor, domestication of wild species and diversification toward development of different breeds. Among the domesticated poultry species, fowl is the first poultry species to be domesticated. The divergence between domestic fowl and its ancestor, Red jungle fowl is presumed to have originated some 8000 years back. Ducks were probably next in sequence (~ 2500 BC), followed by geese (~ 1500 BC), turkey (~ 200 BC) and quail, which is very recent ie. ~ 11th century (Craford 1990). As per taxonomic classification, ducks are in order Anseriformes, while other domesticated poultry species falls in order Galliformes (Howard and Moore 1984). Hence, establishing phylogenetic relationship among these poultry species is important not only for evolutionary aspects but also from genetical aspects which iticludes inter-species divergence, conservation etc. Genetic divergence can be estimated by various biometrical, biochemical and more recently, the molecular genetics approaches. Number of workers have used DNA markers to detect the random variation in genome and to establish genetic relatedness among poultry species (Smith et al. 1996, Sharma ef al. 2001, Joshi 2001). The second strategy is based on amplification of an expressed / function genome and sequence homology comparisons between different genotypes to establish the genetic relatedness among them. The mitochondrial DNA is one of the highly conserved sequences in different species of animals (Antoinette e¢ al, 1995). Sequence variation for mitochondrial genes, specially cytochrome b has been used in estimating genetic relatedness among different avian species (Edwards and Arctander 1997, Bloomer and Crowe 1998, Nishibori et ai. 2001). The 12S rRNA gene is comparatively less exploited gene for such studies. Hence in present study, nucleotide sequence variation in 12S rRNA gene was used to establish the genetic relatedness among domesticated poultry species.Jan - Dec 2005 } MATERIALS AND METHODS DNA extraction Genomic DNA was extracted using phenol-chloroform extraction method from the whole blood collected from brachial vein of red jungle fowl. The DNA was precipitated in presence of high salt concentration by adding chilled absolute alcohol. The DNA was pelleted, washed with 70 % alcohol and dissolved TE buffer. DNA was resolved on 0.8 % agarose gel at 5 V / cm for 2 hrs. The gel was visualized under UV transilluminator and photographed. Lambda DNA digested with Hind III and Eco R I was used as molecular size marker. Amplification and sequencing of 12S rRNA gene Primers for mitochondrial 128 rRNA gene (Forward primer : 5*- CAAACTGGGAT TAGATACCCACTAT-3° and Reverse primer 35’. GAGGGTGACGGGCGGTGTGT-3") were synthesized from MBI Fermentas (M/S Genetix). The DNA. samples were diluted to the concentration of 25-75 ng / ul for PCR. The PCR was set up in 25 yl reaction volume. Amplification conditions used by Girish et al. (2004) were adopted. The amplifications were resolved on 1.4 % agarose. The 100 bp ladder was used as molecular size marker, The amplified ~450 bp 128 rRNA fragment was Table 1. Sequence homology of chicken breeds and avian species 12S tRNA GENE POLYMORPHISM IN RIF AND POULTRY SPECIES purified using commercial kits (Qiagen) and were sequenced at DNA sequencing facility, South Campus, New Delhi using ABI Prism 377 DNA sequencer. ‘Sequence homology comparisons for 12S rRNA gene in different livestock species The comparable nucleotide sequences of 128 rRNA gene from other poultry species, were retrieved from GeneBank nucleotide Sequence database (www.ncbi.nlm.nih.gov/ entrez). The care has been taken to include only those reports, in whom the same set of primers were used (Table 1). These sequences were analysed using the Edit Seq of Laser Gene (DNA STAR Inc.) software. The comparison of sequences from different poultry species was done by Clustral method using Megalign TM software package (DNA STAR). RESULTS AND DISCUSSION Universal primers successfully amplified ~450 bp fragement of 128 rRNA gene in red jungle fowl (Fig. 1). Highly conserved Mitochondrial DNA sequence in different species of animals enable us to design the universal primers for mitochondrial genes, which can amplify corresponding fragments in wide variety of organisms including birds and insects (Prakash ef al. 2000) and also partial sequence of 12S rRNA gene between RIF and other ‘Common name! species Breed / variety ‘Accession No, Red jungle fow! (Gallus gallus) Indian Red jungle fowl 1DQ88S561 (Submitted) Chicken (Gallus g. domesticus) White Leghorn ArB4o444 Quail (Coturix e. Japonica) High BW line AJ-490509 Guinea fowl (Numida meleagris) Pearl AJ.490506 Turkey (Meleagris gallopavo) Small sized White AJ-490508 Duck (Anas Platyrhynchos) Local ‘AJ 490507 27ourta grat 1 26, No. 1,2 m » teaaactggg attagatacc ccactatecs tagccctaaa tetagatace teccatcaca catgtatecy cctyaganct acgageacaa acgcttaasa ctotaaggac ttoyesgtc cecaaaccea cctagaggag cctgttctat aategataat ica ceacgattca cccaaccace ecttgecage acagecteca tacogcegte gccagcccac ctctaatgaa ageacaacag tgageteaat agecectoge taataagace ggtcangges 8 tagectatey ggtgggagaa atgggctaca ttttctaaca a tagaacaaac geasaaggac gtyaascceg cccttageag i gaggatttac gcagtasagt gagateatac ccestaager cactttaaga cggetctgag geacgtacac acoyecogte seccte Fig. 1. Amplification of 128 rRNA gene in red Fig. 2. Nucleotide sequence of 445 bp 125 Jungle fowl, m : 100 bp ladder obviate the requirement for an internal control, which is otherwise used to monitor the success of DNA amplification, PCR assays based on amplification of mitochondrial genes are more sensitive in comparison to single or low copy nuclear DNA targets as vertebrates contain about 1,000-10,000 copies of mitochondrial DNA per cell. It allows the reproducible and reliable amplification of mitocondrial genes even from very small amount of DNA used as a template (Chikuni er al. 1994). Other workers have also reported successful amplification of 128 rRNA gene from different livestock and avian species (Patil et al. 2004). The amplified product from RJF was sequenced using ABI Prism DNA sequencer at DNA Sequencing Facility, University of Delhi, South Campus, New Delhi. Nucleotide sequencing confirmed the size of this 12S rRNA gene in RJF is to be 448 bp. The 5’-3” sequence of the 445 bp fragment so obtained is shown in Fig. 2 Nucleotide sequence comparisons for 12S rRNA sequences from red jungle fowl and other poultry species showed ample variation between them and this variation was used to estimate the genetic similarity and genetic divergence between them (Table 2). Sequence 28 FRNA sequence in red jungle fowl alignment of partial 12S rRNA gene showed that percent genetic similarity between the poultry species ranged from 68.9 to 98.2. While, RIF showed maximum genetic similarity with chicken, least genetic similarity was found with duck. Among other comparisons, except the lower genetic similarity of duck with all other poultry species, the genetic similarity of almost similar magnitude (82.8 to 86.3 %) was observed. While duck showed lower genetic similarity with all other poultry species in general, it was least with turkey (68.9 %), while with other three species, it was more or less of similar magnitude (75.4 — 78.7 %). The genetic divergence between RIF and poultry species ranged from 0.7% — 15.0 % and almost reflected similar trend. Using RAPD markers, much lower genetic similarity was observed between the poultry species, Sharma ef al. (2001) reported genetic similarity of 0.32 to 0.39 (or 32 % - 39 %) between the four poultry species i.e. chicken, quail, guinea fowl and turkey. Similarly, Joshi (2001) reported a very low degree of genetic similarity varying from 0.169 to 0.329 (or 16.9 % - 32.9 %) among the five poultry species including duck, The much lower degree of genetic similarity betweenhe th ad pt all er es ds or ae ist Jan - Dee 2005 } 128 rRNA GENE POLYMORPHISM IN RIF AND POULTRY SPECIES Table 2. Per cent genetic identity and per cent genetic divergence between RIF and other poultry species Species RIF WLH Quail Duck Guinea Fowl Turkey RF - 982 848 774 828 wa WLH 00.7 - 858 784 838 854 Quail 076 076 : 75.4 863 843 Duck 180 150 155 - m2 689 Guinea Fowl 09.2 093 08.5 28 : 87 Turkey 08.9 089 094 148 073 5 poultry species using RAPD markers against very high degree of genetic similarity between same poultry species using sequence comparison for 128 rRNA may be explained as RAPD markers are random and multi locus in nature, hence detects the genetic variability at number of locus simultaneously, while the genetic variation detected by 12S rRNA sequence comparison detects the variation at only one locus. Even at this low level of genetic variability among RJF and poultry species in present study, very high genetic similarity between RIF and chicken (> 99 %) and comparatively much lower Pe cent genetic similarity with other poultry species (< 86 %) is as per expectation. 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