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Develoopment Of A Reliable Microsatellites Maarkers Panel For Parentage Analysis In Cattle Breeds Of Pakistan and Its Validatio Through Cytochrome B Gene Sequencing

By: Tanveer Hussain | Prof. Dr. Masroor Ellahi Babar.
Contributor(s): Dr. Ahmad Ali | Dr. Muhammad Wasim.
Material type: materialTypeLabelBookPublisher: 2013Subject(s): Institute of Biochemistry & BiotechnologyDDC classification: 1597,T Dissertation note: Pakistan posseses enormous Animal Genetic Resource (AnGR) with 36.9 millions of cattle population. The data on genetic fabric of these breed is yet to be documented for their genetic characterization and identification. This work reports first country wide microsatellite markers and cytochrome b gene based genetic characterization of 10 famous cattle breeds of Pakistan. A total of 352 blood samples from unrelated and phenotypically representative of ten native cattle breeds including Bos indicus; Sahiwal, Cholistani, Red Sindhi, Tharparker, Dhanni, Dajal, Lohai, Bhagnari, Achai and Bos indicus x Bos taurus; Nari Master, and an exotic Bos taurus; Holstein Friesian breeds were collected from their respective home tracts, institutional herds and private livestock farms located throughtout the country. These samples were subject to DNA extraction using inorganic method caliberated to same concentration in Molecular Biology and Genomics Laboratory of the Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore Pakistan. A total of 21 microsatellite markers recommended by the programme for the global management of genetic resources (MoDAD) for breed characterization of Food and Agriculture Organization (FAO) of the United Nations and International Society for Animal Genetics (ISAG) were applied. Multiplex PCR were optimized for amplification and were genotyped using ABI Genetic Analyzer 3130 xl using LIZ as size standard. Genotyping results were analyzed using POPGENE and Arlequin ver 3.5 software. The observed and effective number of alleles ranged from 10 (INRA32) to 43 (TGLA126) and 2.3574 (CSSM66) to 15.0019 (BM6526) respectively in all breeds? The observed and expected heterozygosity estimates ranged from 0.0638 (INRA32) to 0.7101 (BM2113) and 0.6510 (INRA32) to 0.9347 (BM6526) respectively in the experimental samples. Mean values for observed and expected heterozygosity was 0.4943 ± 0.1647 and 0.8164 ± 0.0930 respectively. Mean values for Fis, Fit and Fst in all cattle breeds were calculated as 0.2819, 0.3864 and 0.1456 respectively. Average polymorphic information content (PIC) of all microsatellite loci was 0.81 indicating a high degree of informativeness of all microsatellite markers used. It implies that the same set of markers is equally good and could reliably be used for parentage confirmation in Pakistani cattle breeds. The data produced, also showed least degree of genetic difference between Red Sindhi and Tharparker breeds. This may due to mixing of the two breeds for being in close proximity of their home tracts. Fragment mitochondrial cytochrome b gene was also amplified using specific primers through PCR of 130 individuals representing all selected breeds and sequencing was done using ABI Genetic Analyzer 3130 xl. The sequences were aligned and analyzed with CodonCode Alligner 4.0.4 software. The analysis revealed highly degree of sequence conservation in all the Pakistani cattle while documenting changes in only 9 nucleotides from 26 individuals whereas multiple nucleotide changes in 5 locations were shown by more than one individual in the data presented. One polymorphic site was found in nucleotide 318 (T?C) in several breeds of indicine cattle while 2 Lohani and 5 Nari Master individuals showed nucleotide changes specific to taurine cattle. Of all the changes found, only three of them caused changes in the amino acid sequence. The UPGMA tree using MEGA 5.1 showed a clear differentiation between taurine and indicine cattle, except for Nari Master Pakistani cattle showing mitochondrial taurine sequences because it's a cross between Bhagnari (Bos indicus) and Australian Draught Master (Bos taurrus). The estimates of divergence among breeds were also low for most breed pairs, except for Nari Master and Dhanni whereas the overall divergence within Bos indicus or within Bos taurus were also very low (0.002 and 0.003, respectively) but the differences between Bos indicus and Bos taurus were significantly higher (0.014) as should be the case. These results of microsatellite markers have produced a set of information that can be recommended as a reliable marker panel for studies on genetic diversity analysis, parentage confirmation. The cytochrome b data on the other hand not only substantiated genetic diversity analyses but it also proved to be equally good for comparative Phylogenetic analysis of Pakistani cattle breeds and exotic breeds. This work provides most authenticated data and adds a great deal, to already existing information on Pakistani AnGR. This information coupled with prospective data using next generation genetic technologies will assist designing breed improvement focused breeding policies and conservation activities in future.
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Pakistan posseses enormous Animal Genetic Resource (AnGR) with 36.9 millions of cattle population. The data on genetic fabric of these breed is yet to be documented for their genetic characterization and identification. This work reports first country wide microsatellite markers and cytochrome b gene based genetic characterization of 10 famous cattle breeds of Pakistan. A total of 352 blood samples from unrelated and phenotypically representative of ten native cattle breeds including Bos indicus; Sahiwal, Cholistani, Red Sindhi, Tharparker, Dhanni, Dajal, Lohai, Bhagnari, Achai and Bos indicus x Bos taurus; Nari Master, and an exotic Bos taurus; Holstein Friesian breeds were collected from their respective home tracts, institutional herds and private livestock farms located throughtout the country. These samples were subject to DNA extraction using inorganic method caliberated to same concentration in Molecular Biology and Genomics Laboratory of the Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore Pakistan. A total of 21 microsatellite markers recommended by the programme for the global management of genetic resources (MoDAD) for breed characterization of Food and Agriculture Organization (FAO) of the United Nations and International Society for Animal Genetics (ISAG) were applied. Multiplex PCR were optimized for amplification and were genotyped using ABI Genetic Analyzer 3130 xl using LIZ as size standard. Genotyping results were analyzed using POPGENE and Arlequin ver 3.5 software. The observed and effective number of alleles ranged from 10 (INRA32) to 43 (TGLA126) and 2.3574 (CSSM66) to 15.0019 (BM6526) respectively in all breeds? The observed and expected heterozygosity estimates ranged from 0.0638 (INRA32) to 0.7101 (BM2113) and 0.6510 (INRA32) to 0.9347 (BM6526) respectively in the experimental samples. Mean values for observed and expected heterozygosity was 0.4943 ± 0.1647 and 0.8164 ± 0.0930 respectively. Mean values for Fis, Fit and Fst in all cattle breeds were calculated as 0.2819, 0.3864 and 0.1456 respectively. Average polymorphic information content (PIC) of all microsatellite loci was 0.81 indicating a high degree of informativeness of all microsatellite markers used. It implies that the same set of markers is equally good and could reliably be used for parentage confirmation in Pakistani cattle breeds. The data produced, also showed least degree of genetic difference between Red Sindhi and Tharparker breeds. This may due to mixing of the two breeds for being in close proximity of their home tracts.
Fragment mitochondrial cytochrome b gene was also amplified using specific primers through PCR of 130 individuals representing all selected breeds and sequencing was done using ABI Genetic Analyzer 3130 xl. The sequences were aligned and analyzed with CodonCode Alligner 4.0.4 software. The analysis revealed highly degree of sequence conservation in all the Pakistani cattle while documenting changes in only 9 nucleotides from 26 individuals whereas multiple nucleotide changes in 5 locations were shown by more than one individual in the data presented. One polymorphic site was found in nucleotide 318 (T?C) in several breeds of indicine cattle while 2 Lohani and 5 Nari Master individuals showed nucleotide changes specific to taurine cattle. Of all the changes found, only three of them caused changes in the amino acid sequence. The UPGMA tree using MEGA 5.1 showed a clear differentiation between taurine and indicine cattle, except for Nari Master Pakistani cattle showing mitochondrial taurine sequences because it's a cross between Bhagnari (Bos indicus) and Australian Draught Master (Bos taurrus). The estimates of divergence among breeds were also low for most breed pairs, except for Nari Master and Dhanni whereas the overall divergence within Bos indicus or within Bos taurus were also very low (0.002 and 0.003, respectively) but the differences between Bos indicus and Bos taurus were significantly higher (0.014) as should be the case.
These results of microsatellite markers have produced a set of information that can be recommended as a reliable marker panel for studies on genetic diversity analysis, parentage confirmation. The cytochrome b data on the other hand not only substantiated genetic diversity analyses but it also proved to be equally good for comparative Phylogenetic analysis of Pakistani cattle breeds and exotic breeds. This work provides most authenticated data and adds a great deal, to already existing information on Pakistani AnGR. This information coupled with prospective data using next generation genetic technologies will assist designing breed improvement focused breeding policies and conservation activities in future.

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