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Identification Of Single Nucleotide Potymorphisms In Atp Synthase F0 Subunit 6 And Synthase 8 Genes

By: Rizwan Ali | Mr.Tanveer Hussain.
Contributor(s): Prof. Dr. Masroor Ellahi Babar.
Material type: materialTypeLabelBookPublisher: 2013Subject(s): Institute of Biochemistry & BiotechnologyDDC classification: 1570,T Dissertation note: Pakistan is a fertile country regarding Animal Genetic Resource (AnGR), which have more than 1 million camel population belonging to 21 breeds of one humped camel i.e. Camelus dromedarius. Pakistan is the third major camel raising county in the world after Somalia and Sudan. All camel breeds of Pakistan has unique phenotypic traits, however, genetic data is inadequate for their evolutionary and phylogentic study. So to explore and use the genetic potential of dromedary camel, this research work was done. ATP 6 and ATP 8 genes ATP synthase is an enzyme in which oxidative phosphorylation occurs in prokaryotic and eukaryotic cells. The objective of this research was to examine the sequence of ATP 6 and ATP 8 genes in eight camel breeds of Pakistan (Marceeha, Barela, Kachhi, Pahari, Thari, Watni, Kharani, and mix-bred) to find SNPs and to see phylogenetic relationship among them as well as their position while considering already reported camel breeds from all over the world along with other mammalian species in GenBank NCBI. A total of 79 blood samples from eight selected camel breeds of Pakistan, were collected from different government livestock farms and private owners in respective breeding areas of each breed by travelling throughout the country. DNA was extracted and quantified using standard protocols. Specific primers for the selected ATP 6 and ATP 8 genes was designed using primer fox software from reported sequences from the NCBI GenBank (Accession number, JN632608). Primers were optimized and PCR amplification was done on all camel DNA samples. Then all PCR products were processed for sequencing using ABI Prism Genetic Analyzer 3130 xl following standard protocols. Sequence and Phylogenetic analyses All sequences were aligned and analyzed using blast2sequence available on NCBI and CodonCode Aligner. Twenty nine Single Nucleotide Polymorphisms (SNPs) were identified from the aligned 842 bp coding region of ATP 6 and ATP 8 genes. Consensus sequences for eight breeds of Pakistan were used for construction of phylogenetic tree (Neighbor-Joining method) among them using MEGA5.1 software package. The tree indicated high genetic similarity between Mareecha and Pahari camel breeds of Punjab. The Thari, Watni, Kharani and Kachhi breeds of Balochistan province grouped close to each other indicating genetic relatedness among them. Further the phylogenetic trees were constructed for the comparison of Pakistani camel sequences with reported sequences of other camel breeds of the world and different species/ mammals available on GenBank, NCBI. The UPGMA Phylogenetic tree showed the high similarity of all Pakistani camels with Arabian dromedarius camel confirming the dromedarius genetic architecture of Pakistani camels. The two humped (Camelus bactrianus) grouped separately like llama, alpaca (the biological cousins of camel), However both types of camel and llamas were clustered together in one clade while all other mammalian species were grouped together in another clade. However cattle, yak and American bison grouped together, buffalo remained close to cattle. Sheep and goat were also grouped together. Conclusively the phylogenetic tree based on ATP 6 and ATP 8 genes reconfirmed not only the genetic position of Pakistani camel but also the biological/ taxonomic classification of other mammals and species. Significance of research work This work provided the genetic information on eight selected camel breeds of Pakistan and contributed in the existing information in Animal Genetic Resources of Pakistan and helped in exploring the rich genetic structure of our local camel breeds for their effective and meaningful conservation for future generations of Pakistan. This study was just an initial step to explore the genetic worth of Pakistani camel and data produced may act as base line information for other researcher planning to do more research work on camel to get maximum benefits from genetic potential of camel with its unique characteristics. This may also be helpful in designing proper breeding and conservation policies for camel in Pakistan.
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Pakistan is a fertile country regarding Animal Genetic Resource (AnGR), which have more than 1 million camel population belonging to 21 breeds of one humped camel i.e. Camelus dromedarius. Pakistan is the third major camel raising county in the world after Somalia and Sudan. All camel breeds of Pakistan has unique phenotypic traits, however, genetic data is inadequate for their evolutionary and phylogentic study. So to explore and use the genetic potential of dromedary camel, this research work was done.
ATP 6 and ATP 8 genes
ATP synthase is an enzyme in which oxidative phosphorylation occurs in prokaryotic and eukaryotic cells. The objective of this research was to examine the sequence of ATP 6 and ATP 8 genes in eight camel breeds of Pakistan (Marceeha, Barela, Kachhi, Pahari, Thari, Watni, Kharani, and mix-bred) to find SNPs and to see phylogenetic relationship among them as well as their position while considering already reported camel breeds from all over the world along with other mammalian species in GenBank NCBI.
A total of 79 blood samples from eight selected camel breeds of Pakistan, were collected from different government livestock farms and private owners in respective breeding areas of each breed by travelling throughout the country. DNA was extracted and quantified using standard protocols. Specific primers for the selected ATP 6 and ATP 8 genes was designed using primer fox software from reported sequences from the NCBI GenBank (Accession number, JN632608). Primers were optimized and PCR amplification was done on all camel DNA samples. Then all PCR products were processed for sequencing using ABI Prism Genetic Analyzer 3130 xl following standard protocols.
Sequence and Phylogenetic analyses
All sequences were aligned and analyzed using blast2sequence available on NCBI and CodonCode Aligner. Twenty nine Single Nucleotide Polymorphisms (SNPs) were identified from the aligned 842 bp coding region of ATP 6 and ATP 8 genes. Consensus sequences for eight breeds of Pakistan were used for construction of phylogenetic tree (Neighbor-Joining method) among them using MEGA5.1 software package. The tree indicated high genetic similarity between Mareecha and Pahari camel breeds of Punjab. The Thari, Watni, Kharani and Kachhi breeds of Balochistan province grouped close to each other indicating genetic relatedness among them. Further the phylogenetic trees were constructed for the comparison of Pakistani camel sequences with reported sequences of other camel breeds of the world and different species/ mammals available on GenBank, NCBI. The UPGMA Phylogenetic tree showed the high similarity of all Pakistani camels with Arabian dromedarius camel confirming the dromedarius genetic architecture of Pakistani camels. The two humped (Camelus bactrianus) grouped separately like llama, alpaca (the biological cousins of camel), However both types of camel and llamas were clustered together in one clade while all other mammalian species were grouped together in another clade. However cattle, yak and American bison grouped together, buffalo remained close to cattle. Sheep and goat were also grouped together. Conclusively the phylogenetic tree based on ATP 6 and ATP 8 genes reconfirmed not only the genetic position of Pakistani camel but also the biological/ taxonomic classification of other mammals and species.


Significance of research work
This work provided the genetic information on eight selected camel breeds of Pakistan and contributed in the existing information in Animal Genetic Resources of Pakistan and helped in exploring the rich genetic structure of our local camel breeds for their effective and meaningful conservation for future generations of Pakistan. This study was just an initial step to explore the genetic worth of Pakistani camel and data produced may act as base line information for other researcher planning to do more research work on camel to get maximum benefits from genetic potential of camel with its unique characteristics. This may also be helpful in designing proper breeding and conservation policies for camel in Pakistan.

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