Characterization of Myostatin Gene and Identification of SNPs for Diversity Analysis

S. T. Bharani Kumar 1, 2 , Neeraj Dilbaghi ² , S P S Ahlawat³, Bina Mishra ¹ , M. S. Tantia ¹ * an R. K. Vijh ²

1National Bureau of Animal Genetic Resources, Karnal 132001, India.

2Guru Jambheshwar University of Science and Technology, Hisar

3Indian Veterinary Research Institute, Izatnagar.

* For corresepondance - mstantia @yahoo.com

Current Trends in Biotechnology and Pharmacy, Vol.1 (1) 70-78 (2007) ISSN:0973 - 8916

Abstract

In this present study we have sequenced the complete Myostatin gene with 3 exons, twointrons, promoter region, 5’ and 3’ UTR region.We sequenced the entire gene by designing a setof 20 primers. Ten individuals from 5 breeds ofIndigenous chicken were sequenced and thesequences obtained were aligned to develop aconsensus sequence. We compared the sequencesobtained among themselves, with Red JungleFowl and Chinese fowl. We estimated threegenetic distances and constructed thephylogenetic tree using NJ method. Theindigenous chicken joined together first followedby Red Jungle Fowl while the Chinese fowl wasdistinct. The indigenous gene sequence wassimilar among the various chicken breeds andmight have been evolved from RJF following aseparate course than the evolution of Chinesechicken.

Introduction

Myostatin (GDF-8) is an important candidategene for growth and development of domesticanimals and poultry. It is a member of TGF-âsuperfamily of secreted growth anddifferentiation factors. It is a negative regulatorof skeletal muscle growth and is essential ofproper regulation of skeletal muscle mass (1). The gene has been studied widely in cattle (2, 3,4), buffalo (5), mice (6) and chicken (7,8). Thisgene has been reported to provide geneticevidence of role of SNPs in myostatin gene inregulation of adipose growth and musculaturein chicken (8). Myostatin SNPs have beenreported to occur with different gene frequenciesamong chicken breeds⁷.

The comparative analysis of broiler, layer andsilkie fowl genome with Red Jungle fowl (RJF)have revealed 3.2 million SNPs which are nowavailable in public domain (Chicken VDDatabase, 9). The indigenous chicken aremorphologically of two distinguished types, thegame birds which have a very distinctive bodyconformation and well developed musculaturecompared to other normal layer type birds.

The present study was carried out to sequencethe myostatin gene in five indigenous chickenpopulations and comparing the sequences amongthemselves to develop a consensus sequence ofindigenous chicken. These included the normalbirds as well as game birds. Aseel and Danki arethe game birds and have well developedmusculature with aggressive behaviour (10).These birds are found in junction of AndhraPradesh, Jharkand and Orissa. Daothigir birdsare found in the western Assam bordering WestBengal and are reared by Bodo tribals. Punjab brown are normal birds of Punjab and Haryanaof North India while Chittagong birds inhabit andMeghalaya bordering Bangladesh. The fiveIndian chicken populations, RJF and Chinesechicken have been compared for identifying thevariations. The purpose of the present study wasto report variations among the chicken breeds ofIndia and to reconstruct phylogeny with otherchicken populations and RJF.

Material and Methods

Samples: Two samples each from the five populations of indigenous chicken were utilized.Genomic DNA was extracted from 0.5 ml ofblood collected from wing vein in heparinisedvacuutiners. The DNA was isolated using thestandard laboratory protocols (11).

Sequencing of Myostatin gene: We designed twenty pairs of overlapping primers (Fig 1) for complete chicken myostatin gene sequences(Table 1) available in NCBI database(AF346599) using primer3 software. The PCRconditions were standardized for eacholigonucleotide primer pair. Amplified PCRproducts were purified by treating withExonuclease and Alkaline phosphatase (0.5 uniteach) and incubating at 37C for 2 hrs followedby inactivation at 850C for 15 minutes. PurifiedPCR products were sequenced using Big Dyeterminator chemistry on 3100-Avant (AppliedBiosystems) automated DNA sequencer.

Sequence Analysis:Myostatin gene sequencefrom the five breeds of chicken were alignedusing Seqscape version 3.0 software (AppliedBiosystems, USA). The variations among theindigenous breeds were recorded. The consensussequence was obtained for the indigenous breedsand was compared to Chinese native chicken(AF346599) and sequence of the gene of RedJungle fowl (Ensembl release Ver 4.6 Gallusgallus) (12). The sequence of the indigenousbreeds, Chinese fowl and RJF were subjected to multiple alignment using ClustalW (13) andgenetic distances based on nucleotide differenceswere obtained using Phylip software moduleDNADist (14). The output was utilised for theconstruction of Neighbor joining tree (15) andviewed using Treeview (16). We utilized themaximum composite method, Kimura-2parameter model (using MEGA software, 17) andDNA dist (Phylip software) for estimating thedistances.

Results and Discussion

The consensus sequence of these ten individualswas prepared covering the entire myostatin gene with promoter and 5’ UTR region. The consensussequence was submitted to NCBI database alongwith variations observed (DQ912835). The totalnucleotide bases were 8175bp and consisted ofpromoter region (1-1433), 5’UTR (1434-1550),Exon1 (1434-1923), Exon2 (4015-4388) andExon3 (6664-8175) with 3’UTR (7045-8175).The intervening sequences were for the twointrons.

The comparison of the sequences obtained withthe reference sequence AF346599 revealedseveral changes (Table 2). The variationsobserved included 17 deletions, 3 insertions of2(CT), 3(ACC) and 11(TTAGTGTTTTT) basesat 8527, 7561 and 4923 respectively. The totalnumber of transitions and transversionscompared to reference sequence were 15 and 17respectively. At position 1784 there was atransition in Exon1 which was synonymous withno change in aminoacid. There was anotherchange non synonymous in the Exon3 at position7414 and 7415 (both transitions of pyrimidine)which lead to single aminoacid change fromProline to Serine. These changes were peculiarto indigenous chicken populations.

The comparison of GDF-8 gene sequenceobtained in the present study with the genesequence of Red Jungle fowl available in Ensembl database revealed few variations (Table3). The changes were T1280C in promoterregion, C1468A and G1521A in 5’UTR regionand G1610A and C1745G in Exon1. Thevariations among the indigenous chickenpopulations revealed 44 SNPs of which 31 weretransitions and 9 transversions (Table 4). TheSNP distribution is as follows- twenty three inpromoter (Fig 2), five in exon 1 (Synonymous),seven in intron 1, eight in intron 2 and one in3’UTR. Four deletions were observed in thepromoter region.

The evolutionary history of the five indigenouschicken populations, Red Jungle fowl andChinese fowl was inferred using the Neighbor-Joining method. The distance values obtainedfrom maximum composite method are given intable 5. Among the fowl populations, the geneticdistances were maximum for the Chinese fowlwhile the indigenous chicken genetic distancevalues were quite small followed by geneticdistance from the Red Jungle Fowl. Thus thegenetic distances reveal that the indigenouschicken populations have diverged separatelyfrom the Red jungle fowl and all the breeds haveevolved by distance and isolation amongthemselves while Chinese fowl had taken aseparate course and evolved differently than theindigenous chicken (Fig 3). The phylogenetictree reconstructed was same for the DNAdist,Kimura-2-parameter model and maximumcomposite method.

Conclusion

The Myostatin gene sequences provide evidencethat the Aseel and Dunki are game birds and closeto one another in the phylogenetic tree. Daothigirjoin Aseel at the first node. Daothigir are normallayer birds but breed in close vicinity of thebreeding tract of game birds which extend allalong the coastal regions of India intoBangladesh. Chittagong and Punjab Brown birdsare layers and have their breeding areas adjoining to that of Red Jungle fowl which exists in wildall along the Himalayan belt from northern tonorth eastern India. Since the indigenous chickenare continuously distributed with no clear cutdistinction between the areas inhabited by RedJungle fowl, game birds and other layer birdsand such a pattern is also revealed at the genesequence level (Myostatin gene in the presentcase). Aseel and Danki come close to one anotheras both are selected for the game characteristicsand are reared by the tribals.

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Table 1: List of primers, sequences and their product size used to amplify the complete myostatin gene

Table 2:variations observed in Indigenous domestic chicken compared to reference sequence

Table 3:Variations observed between Indian indigenous Chicken and RJF sequences obtained from Ensembl gene Browser

Table 4: SNPs in myostatin gene among five different populations

Table 5: DNA dist (above diagonal) and inter-population difference (below diagonal).

Fig 1:Overlapping primers designed for Myostatin (GDF-8) gene using primer3 software

Fig 2:SNPs detected in promoter region (G840A and T850C) from alignment of sequencesusing SeqScape ver 3.0

Fig 3: The optimal tree with the sum of branch length = 0.00350199 was constructed. The treedepicted is drawn to scale, with branch lengths in the same units as those of the evolutionarydistances used to infer the phylogenetic tree. The evolutionary distances were computed using theMaximum Composite Likelihood method and are in the units of the number of base substitutionsper site. The NJ algorithm was used for the construction of the tree. All the positions containinggaps and missing data were eliminated from the dataset and there were 6702 positions in the finaldataset used to construct the tree.