Parentage Analysis And Breed Characterization Of Dogs By Microsatellite Markers
Material type: Book ; Format:
Publisher: 1990 Dissertation note: Pakistan has vast population of dogs belonging to different breeds. Most of the dogs have no pedigree record which is a great threat to conservation of different breeds. No study on DNA fingerprinting of dogs has been conducted in Pakistan. DNA fingerprinting of dogs is necessary to overcome the problems like forensic cases, sale & purchase, individual identity in case of fertilization by more than one male and ownership disputes. Microsatellite markers have been proved as an efficient and powerful tool for parentage testing and breed characterization of dogs. In this study, a panel of microsatellite markers, having high polymorphism information content (PlC) values, was developed. Blood samples were taken from cephalic vein of two breeds of dogs (German shepherd and Labrador retriever). DNA was extracted by Inorganic method. Primers of microsatellite markers were optimized for successful amplification conditions in the Bio-Rad thermocycler. Multiplex PCR was performed, for amplification of these microsatellite markers on 46 samples belonging to 20 families. Genotyping analysis was performed for the PCR products of microsatellite markers on non denaturing polyacrylamide gel. These results were analyzed statistically software "POPGENE 3.3 and POWER STAT". Allele frequency, heterozygosity, homozygosity, polymorphism information content (PlC), power of discrimination and power of exclusion of all microsatellite markers were calculated. Average power of discrimination among non parents, average hetrozygosity, average observed homozygosity and average polymorphism information content (PlC) value for all alleles was 0.809, 0.6345, 0.2913 and 0.724 respectively. Moreover combined power of exclusion reached a significant value of 0.9998. Almost all of the microsatellite markers showed significant variations in both German shepherd and Labrador retriever breeds. Microsatellite "REN41D2Ob" showed maximum variation i.e. 17 alleles and microsatellite"REN49F22b" showed the least variation among all microsatellite markers i.e. 4 alleles. Genotyping results of microsatellite markers were clearly different for two different breeds showing a distinct genetic distance between German shepherd and Labrador retriever breeds.
Results of this study lead to development of a panel of microsatellite markers which can be used for parentage analysis and breed characterization of dogs. This was a preliminary study on dogs in Pakistan. This facility can be provided on commercial basis to pet owners and kennel clubs. Moreover this study can become the basis for further research investigations in canines in Pakistan.
Availability: Items available for loan: UVAS Library [ Call number: 1090,T] (1).
Identification And Molecular Characterization Of Shiga Toxin Producing
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Publisher: 2011 Dissertation note: Escherichia coli is normal inhabitant of all the animals and human beings. Its non-sorbitol fermenting biotype (SNF) was detectable in buffalo (90 percent), cattle (80 percent) or rarely in sheep (20 percent) and goat (30 percent). However, SNF E. coli were un-detectable in droppings of rural chickens and feces of donkeys. The SNF E. coli was detected in 100, 92, 71 and 80 percent of the market milk samples and 100, 83, 83 and 53 percent beef samples from Multan, Sandha, Wagha and Sheikhupura Roads, of Lahore city, respectively. However, SNF E. coli was not detected from freshly aseptically collected milk and beef samples but was detectable in over all 96 percent of market raw milk and 82 percent market beef samples. White colored colonies of each positive sample on Sorbitol MacConkey's Agar (SMA) contained gram negative, rods with round ends non spore former. Each of the isolates was sorbitol non fermenter, lactose fermenter, indole positive, Methyle Red positive, Voges Prauskaur negative and citrate negative. However, each of such isolates showed green metallic sheen on Eosin Methylene Blue (EMB) agar.
Each of the isolate was further characterized using polymerase chain reaction (PCR). Bacterial DNA was extracted easily by boiling method when the isolates were grown on Sorbitol MacConkey's agar or EMB agar at 370C for 12-24 hours. The DNA was recovered when the culture was boiled for 5-10 minutes. The DNA of each sample remained stable on storage at 40C for 48 hours or at -200C for 7 days. The isolated DNA (100 samples) when amplified using universal, Stx1, Stx2 and O157 specific primers showed that 82 percent samples were positive for universal primers, 50 percent for O157, 60 % for Stx1 and 51 percent for Stx2. The filtrate of each isolate when diluted as 1:10 dilution and sterilized by filtration induced cyto-pathogenic effect (CPE) on Vero cell line.
It is concluded that SNF E. coli O157 normally exists in intestinal tract of buffalo, cattle, sheep and goat. Counts of SNF E. coli O157 were higher in milk samples as compared to beef samples. More than 80 percent samples of milk or beef were contaminated with SNF E. coli O157. Feces of the animals are presumably main source of SNF E. coli contamination of raw milk and beef. PCR is a quick, reliable, and sensitive technique for confirmation of SNF E. coli O157 in the samples.
Availability: Items available for loan: UVAS Library [ Call number: 1221,T] (1).
Identification Of Single Nucleotide Potymorphisms In Atp Synthase F0 Subunit 6 And Synthase 8 Genes
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Publisher: 2013 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.
Availability: Items available for loan: UVAS Library [ Call number: 1570,T] (1).