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Author: ANILKUMAR, K. × End date: 2009 × Start date: 2000 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    DELINEATION OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS IN CROSSBRED CATTLE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2003) ANILKUMAR, K.; RagiTunandanan, K. V.
    A study was conducted to delineate the Random Amplitied PoKmorphic DNA markers of crossbred cattle of Kerala. DNA was isolated from whole blood, fresh semen or frozen semen samples for this study. Three panels of DNA samples were used in this stud)'. The)' were DNA samples of 84 unrelated crossbred cattle, which was used for assessing the RAPD polymrophic patterns. DNA samples from 52 offspring, dam and sire combinations for assessing the possibility of using the technique in parentage identification and DNA samples ot 108 dwarf cattle for microsatellite analysis. The average yields of DNA obtained from whole blood was 256 ± 2.12|ig/ 5 ml. fresh semen was 241.23 ± 8.42pg/ 400 million sperms and trom frozen semen was 91.32 ± 6.01 pg/ 0.25 ml straw: The optical density ratio calculated as an indicator of purity of the sample were 1.72 ± 0.14. 1.81 ± 0.26 and 1.61 ± 0.34 for the DNA obtained from whole blood, frozen semen and fresh semen respectively. Twenty random oligonucleotide primers from Operon kit A and six custom svnthesised primers were used for the stud). Based on intensit). clarity and polymorphism of bands. 19 primers were selected for amplification of DNA samples of crossbred cattle. Number of bands obtained from different primers ranged from 2 to 16. The acerage number of bands produced by different primers ranged from 3.78 ± 0.15 in OPA 4 to 8.15 ± 0.25 in ILO1127. The size of amplified products ranged from -230 base pairs to -3580 base pairs. Percentage of polymorphism represented by indi\ idual primer caried from 66.66 to 100 in different groups. Se\en primers namely 0PA2. OPA 4. OPA 17. OPA 18. OPA 20. ILO 1127 and ILO 876 yielded 100% polymorphic bands. Frequencies of the bands and their allelic frequencies were worked out. The largest average number of bands was produced by the primer ILO 1127 ( 8.15 ± 0.25)-and the lowest average number of bands was observed for the primer OPA 4 (3.78 ±0.15). The bands having frequency less than 0.25 were classified as rare bands. The primer OPA 15 produced 7 rare bands, the primer OPA 2 and OPA 19 five bands each, the primers for OPAl. 0PA4. OPA 8. OPA 9.OPA 16 and ILO 1127 four each and primers 0PA7. OPA 14. OPA 17 and ILO 876 three each.ILO 526oduced two rare bands, four primers namely OPA 10. OPA 12. OPA 18 and OPA 20 produced one rare band each . and no rare bands were observed for the primer Gl. Twelve RAPD primers were selected to study 52 offspring, dam and sire combinations. Non parent bands were observed in offspring to the extent of 2 to 13.6%. Three of these primers nameh' OPA 14. OPA 4 and 01 did not produce any non parent bands in offspring. It was concluded that due to presence of the non parent bands in offspring. RAPD-PCR technique cannot be the method of choice for parentage verification. Phenol chloroform procedure was used to isolate DNA from whole blood of 24 Vechur. 25 Highrange dwarf. 24 Vatakara and 35 Kasargode animals for microsatellite studies. The average yield of DNA obtained per 5 ml ot blood samples was 210.1 ±9.4 pg with the ratio of optical density at 260 nm and 280 nm as 1.86 ± 0.23. PGR conditions for the four microsatellite loci namely DRB3. ETH 131. HEL 6 and FSHp were standardised. The size of the alleles ranged from 176 to 236 bp for DRB3 locus. 160 to 184 bp in ETH 131 locus and 263 to 295 base pairs in HEL-6 locus. The number of alleles identified in different loci were 21. 14 and 13 respectively for DRB3. ETHI31 and HEL 6 loci. The PIC \ alue of the primers, direct count heterozygosity and unbiased heterozygosit) were worked Ill out. The identification ot new alleles in this stud) was attributed to the tact that dwarf cattle of Kerala are Bos indiciis . The possibilit> of using the microsatellite marker analysis for genetic characterization of dwarf cattle ot Kerala and their phylogenic studies are indicated in this work. Parentage \eritication tacilities can be established, where this technique can be employed in disputed cases.